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This is the PhD thesis of Michael J Cox, published by the University of Warwick in 2005. The work covers analysis of marine methyl halide-utilising bacteria and attempts to associate the presence and diversity of these organisms with the presence and abundance of methyl bromide in seawater. Methyl bromide is a trace atmospheric and marine gas that is involved in ozone depletion. It has a natural biogeochemical cycle, but is also produced synthetically for use as a fumigant.
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i
Marine Methyl Halide-Utilising Bacteria
A thesis submitted by
Michael J Cox BSc (Hons)
to
The Department of Biological Sciences in
fulfilment of the requirements for the degree of
Doctor of Philosophy
July, 2005
University of Warwick
Coventry, UK
ii
Dedicated to the girl who sang the blues.
iii
Table of contents Title Page i Table of contents iii List of Figures iv List of Tables vi Abbreviations viii Acknowledgements xi Declaration xii Publications xiii Abstract xiv Chapter 1: Introduction 1 Chapter 2: Materials and Methods 35 Chapter 3: Measurement of Methyl Bromide 58 Chapter 4: Enrichment and Isolation of Methyl Bromide-Utilising Bacteria 83 Chapter 5: Methanol Dehydrogenase as a Functional Genetic Marker 100 Chapter 6: Diversity of cmuA in Marine Environments: Clone library and TRFLP analysis 119 Chapter 7: Leisingera methylohalidivorans strain MB2 and attempts to identify cmuA 144 Chapter 8: Synopsis, Discussion and Future Work 153 Bibliography 164 Appendices 181
iv
List of Figures Chapter 1 Figure 1.1 Global methyl bromide budget 5 Figure 1.2 Metabolism of C1 compounds by aerobic methylotrophic
bacteria 10
Figure 1.3 Pathway of CH3Cl degradation in Methylobacterium chloromethanicum CM4
14
Figure 1.4 Comparison of cmu gene clusters sequenced to date 22 Figure 1.5 Maximum likelihood tree of 16S rRNA sequences isolated
strains of CH3X utilisers 24
Figure 1.6 Location of sampling station L4 in the English Channel 31 Figure 1.7 Arabian Sea AMBITION cruise track 33 Chapter 3 Figure 3.1 Diagrammatic representation of the Electron Capture
Detector (ECD) 62
Figure 3.2 Purge apparatus for GC ECD ‘system two’ 64 Figure 3.3 Gas purifier system for the carrier, make-up, sparge and
Nafion counter flow gases of GC ‘system two’ 66
Figure 3.4 Diagram of GC ECD System two 68 Figure 3.5 L4 CH3Br measurements 73 Figure 3.6 Phytoplankton abundance and CH3Br concentration at L4 74 Figure 3.7 Pigment concentrations during the AMBITION cruise 78 Chapter 4 Figure 4.1 Chemical equation for complete oxidation of CH3Br 85 Figure 4.2 Oxidation of CH3Br by four enrichments 91 Chapter 5 Figure 5.1 a α subunit of methanol dehydrogenase with coordinated
PQQ and Ca2+ 102
Figure 5.1b β subunit of methanol dehydrogenase. 102 Figure 5.1c α2β2 structure of methanol dehydrogenase. 102 Figure 5.2 Alignments mxaF sequences used for PCR primer design 108 Figure 5.3 mxaF PCR of a range of environmental samples 112 Figure 5.4 Phylogenetic analysis of mxaF sequences of strains used in
PCR primer design 115
Chapter 6 Figure 6.1 Pathway of CH3Cl degradation in Methylobacterium
chloromethanicum CM4 (as figure 1.3) 120
Figure 6.2 Comparison of cmu gene clusters sequenced to date (as Figure 1.4)
122
Figure 6.3 Example EcoRI/DdeI RFLP digest of cmuA clones from enrichment L4.1
125
Figure 6.4 cmuAF802/cmuAR1609 PCR products from CH3Br enrichments
126
Figure 6.5 Phylogenetic analysis of all cmuA sequences; parsimony analysis and clade assignment
131
Figure 6.6 Phylogenetic analysis of selected cmuA sequences; maximum-likelihood analysis
132
Figure 6.7 BsiYI TRFLP pattern of clone PMLSW6 (AJ810829) 139 Chapter 7 Figure 7.1 Alignment of cmuA sequences with primer cmuAF802 146
v
Figure 7.2 Alignment of cmuA sequences with primer cmuAR1609 147 Figure 7.3 Alignment of cmuA sequences with primer cmuAR1244 147 Figure 7.4 3D-views of a four helix bundle corrinoid-binding domain
with bound cobalamine 148
Figure 7.5 Alignment of cmuA sequences with primer cmuAR1352 149 Figure 7.6 Autoradiograph of L. methylohalidivorans Southern
analysis 151
Figure 7.7 L. methylohalidivorans MB2 restriction digested DNA samples prior to Southern hybridisation analysis
151
Appendices Figure A.2 Physicochemical data from the Arabian Sea AMBITION
cruise 182
Figure A.3 Productivity data from the Arabian Sea AMBITION cruise 184 Figure A.4 Microorganism abundance data from the Arabian Sea
AMBITION cruise 186
Figure C.1 Excel spreadsheet for Henry’s Law calculations 199
vi
List of Tables Chapter 2 Table 2.1 Bacterial and algal strains used in this study 37 Table 2.2 Genomic DNA extracts used in this study 38 Table 2.3 Cruise enrichment carbon sources 45 Table 2.4 Selected media concentrations of CH3X in various culture
formats 46
Table 2.5 PCR Primers used in this study 52 Chapter 3 Table 3.1 Detection limits of a selection of common detectors used in
gas chromatography 61
Table 3.2 Marine phytoplankton demonstrated to produce CH3Br in laboratory cultures
76
Table 3.3 Linking pigment presence with classes of phytoplankton 77 Chapter 4 Table 4.1 Cruise enrichment carbon sources 86 Table 4.2 Turbidity estimation of Arabian Sea cruise enrichments 87 Table 4.3 Arabian Sea enrichments positive for CH3X utilisation 88 Table 4.4 Total CH3X consumed by enrichments 90 Table 4.5 Emiliania huxleyi culture axenicity 92 Table 4.6 Strains isolated from CH3Br enrichments of Arabian Sea
samples 95
Table 4.7 Amounts of substrate used in O2 electrode studies 96 Table 4.8 Substrate affinity and maximum oxidation rate of H.
chloromethanicum CM2 with CH3X 97
Chapter 5 Table 5.1 mxaF sequences used for PCR primer development 106 Table 5.2 mxaF PCR primers used in this study 107 Table 5.3 Expected product sizes for each of the combinations of
primer and results of the first trials 110
Table 5.4 Genomic DNA samples used for testing efficacy of primer pairs
113
Chapter 6 Table 6.1 OTUs from library 25, the cmuA clone library from
enrichment 249 126
Table 6.2 OTUs from library 27, the cmuA clone library from enrichment PE2
127
Table 6.3 OTUs from library 9, the cmuA clone library from enrichment PE2, amplified with primers cmuAF229/cmuAR1609
128
Table 6.4 Effective sample volumes for 1 µl volumes of SAP sample DNA extracts
129
Table 6.5 OTU assignment of cmuA sequences from SAP sample clone libraries
130
Table 6.6 TRF sizes of known cmuA sequences 137 Table 6.7 Absorption and emission maxima of fluorophores for
TRFLP analysis 139
Table 6.8 AMBITION samples analysed by cmuA PCR 140 Table 6.9 Celtic Sea samples 141
vii
Appendices Table A.1 Location of casts used for data analyses of the Arabian Sea
AMBITION cruise 181
Table B.1 List of Arabian Sea AMBTION cruise DNA samples 187 Table B.2 List of Arabian Sea AMBTION enrichments 191 Table D.1 Clade affiliation of cmuA TRFs based on in silico analysis
of database cmuA sequences 120
viii
Abbreviations
A adenine
AMO ammonia monooxygenase
ANMS ammonium nitrate mineral salts
ATP adenosine-5’-triphosphate
BAC bacterial artificial chromosome
bp base pair
BLAST basic local alignment search tool
BSA bovine serum albumin
C cytosine
CFC chlorofluorocarbon
CH3Br methyl bromide (bromomethane)
CH3Cl methyl chloride (chloromethane)
CH3I methyl iodide (iodomethane)
CH3X methyl halide (halomethane)
CODEHOP consensus-degenerate hybrid oligonucleotide primers
CoM coenzyme M
CTD conductivity, temperature and depth
Da Dalton
DMA dimethylamine
DMSO dimethyl sulfoxide
DMSP dimethyl sulfoniopropionate
DNA 3’-deoxyribonucleic acid
DGGE denaturing gradient gel electrophoresis
ECD electron capture detection
ix
EDTA ethylenediaminetetraacetic acid
FID flame ionisation detection
FISH fluorescence in situ hybridisation
G guanine
GC gas chromatography
H4F tetrahydrofolate
H4MPT tetrahyhdromethanopterin
ID identity
kb kilobase
kDa kiloDalton
LB Luria Bertani
MAMS marine ammonium mineral salts
MAMSTY marine ammonium mineral salts with tryptone and yeast
extract
MDH methanol dehydrogenase
MMA monomethylamine
mRNA messenger ribonucleic acid
NMS nitrate mineral salts
OD optical density
ORF open reading frame
OTU operational taxonomic unit
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
pMMO particulate methane monooxygenase
ppmv parts per million by volume
x
pptv parts per trillion by volume
PQQ pyrrolo-quinoline quinone
RFLP restriction fragment length polymorphism
RNA ribonucleic acid
rRNA ribosomal ribonucleic acid
RuMP ribulose monophosphate
SDS sodium dodecyl sulfate
SIP stable isotope probing
sMMO soluble methane monooxygenase
T thymine
TBE Tris-borate EDTA
TE Tris-EDTA
TMA trimethylamine
Tris Tris-(hydroxymethyl)-aminoethane
TRFLP terminal restriction fragment length polymorphism
analysis
U uracil
uv ultraviolet
v/v volume to volume
w/v weight to volume
xi
Acknowledgements
My first thanks must go to my three supervisors, Prof. Colin Murrell, Dr. Ian McDonald and Dr. Phil Nightingale who were fantastic supervisors and an excellent team despite being geographically as far-flung as you can be. Thank you very much for your help, advice and patience. I have worked with a large number of people in Warwick, Plymouth and the middle of the Arabian Sea and I would like to thank everyone I have ever asked for advice, equipment or support, there was not a single occasion when any of these were refused without good reason. At Warwick special thanks must go to the methyl halide team, now sadly disbanded; Hendrik Schäfer, a fantastic friend and unofficially supervisor number four, Elena Borodina, my coordinated dancing partner, and Karen Warner, whose selectively embarrassing taste in music matched my own. Stefan Radajewski and Johannes Scholten were endless sources of knowledge on molecular biology and thermodynamics respectively that I hope I have learnt from. I would also like to thank Gez Chapman for all his help and Julie, Moh, Ju-Ling, Marc, Simon, Matt, Jo, Hanif, Andi, and everyone else in Micro I for making it a fantastic place to work . Thank you to Helen Bird, and the ladies of the Molecular Biology Service for help and advice, particularly with TRFLP. At PML the Biogas and Tracer Group were extremely welcoming and patient, if I can tell one end of a spanner from the other it’s down to you . Particular thanks to Malcolm Liddicoat for his help and advice, John Wood for egg custard tarts, and to Dr. Laura Goldson for laughing at my jokes. I would also like to thank Norman Reville and the crews of RVS Squilla, RVS Sepia and RVS Plymouth Quest for getting me to L4 and back in one piece, and for making the experience enjoyable. The AMBITION cruise was a baptism of fire at the very beginning of my PhD; I would like to thank the Captain and crew of RRS Charles Darwin and all the participants. I would especially like to thank Dr. Malcolm Woodward, Dr. Andy Rees, Dr. Glen Tarran and Denise Cummings who took me under their wing from the start, and Dr. Nick Fuller, Dr. Clare Bird and Dr. Karen Orcutt who made it all the more fun. I have never consumed so much gin and hope never to again. I would also like to thank my Mum, Dad and brother for many things, but in particular for taking me Wembury beach, letting me poke about in rock pools for hours, and for being interested in every unremarkable shell, stone or bit of seaweed I ever showed you. Who’d have thought someone would pay you to do it? Finally thank you to Karen who suffered at least as much as me whenever an experiment went wrong and had to endure every whinge. You are so much more patient than me and I love you very much.
xii
Declaration
The work present in this thesis is original work conducted by myself unless stated otherwise, under the supervision of Prof. Colin Murrell and Dr. Ian McDonald at the University of Warwick and Dr. Phil Nightingale at Plymouth Marine Laboratory. The measurements of methyl bromide at L4 in Chapter 3 were carried out by Malcolm Liddicoat, Plymouth Marine Laboratory. All sources of information have been acknowledged by reference. None of this work has been used in any previous application for a degree.
xiii
Publications Borodina, E., Cox, M. J., McDonald, I. R., and Murrell, J. C. (2005) Use of DNA-
stable isotope probing and functional gene probes to investigate the diversity of
methyl chloride-utilizing bacteria in soil. Environmental Microbiology. 7(9) 1318-
1328.
McDonald, I. R., Kampfer, P., Topp, E., Warner, K. L., Cox, M. J., Connell
Hancock, T. L., Miller, L. G., Larkin, M. J., Ducrocq, V., Coulter, C., Harper, D.
B., Murrell, J. C. & Oremland, R. S. (2005) Aminobacter ciceronei sp. nov. and
Aminobacter lissarensis sp. nov., isolated from various terrestrial environments.
International Journal of Systematic and Evolutionary Microbiology. 55(5) 1827-
1832.
xiv
Abstract
Methyl bromide is a potent ozone-depleting atmospheric trace gas with both natural and anthropogenic sources, and a complex natural cycle. The sources and sinks of methyl bromide are numerous and are currently unbalanced. The oceans are both a source and a sink of methyl bromide, but believed to be a net sink. Chemical degradation rates of methyl bromide in the oceans are too slow to account for the observed loss of this methyl halide and degradation is thought to be due to oxidation by marine bacteria. A number of bacteria have been isolated that are able to use methyl halides, including methyl bromide, as sole source of carbon and energy. The gene cmuA encodes a methyltransferase/corrinoid-binding protein that has been shown to catalyse the first step in a methyl chloride and methyl bromide utilisation pathway. Components of this pathway have been shown to be present in the majority of methyl halide-utilising bacteria that have been isolated and cmuA has been used as functional genetic marker of methyl halide utilisation by bacteria. In this study, the presence and diversity of marine methyl bromide-utilising bacteria was investigated by enrichment, isolation and molecular biological methods, alongside the measurement of methyl bromide in seawater samples by a sensitive gas chromatography with electron capture (GC ECD) technique. Methyl bromide enrichments of seawater samples from the Arabian Sea and from L4, a sampling station in the English Channel off the coast of Plymouth demonstrated oxidation of methyl bromide. Measurements of methyl bromide by GC ECD at L4 suggested rapid biological removal of methyl bromide dissolved in the water column. Isolation of methyl bromide-utilising bacteria was attempted and amplification of marine bacterial cmuA sequences achieved from a number of enrichments. These were cloned and sequenced and their diversity analysed, resulting in the identification of three clades of marine cmuA sequences. DNA extracted from large volumes of Arabian Sea seawater from a cruise track that covered 11 sampling stations mainly along the 67 oE meridian was also PCR amplified using cmuA specific PCR primers. PCR products were cloned, dereplicated and sequenced and phylogenetic analysis assigned the sequences to the same three clades of cmuA identified from the enrichments. A shift from one clade to another could be seen between the oligotrophic station one and the more eutrophic stations four and nine of the Arabian Sea cruise track. A rapid microbial assemblage fingerprinting technique was developed for use with cmuA. A further functional genetic marker, mxaF, encoding the large subunit of the methanol dehydrogenase of methylotrophs and methanotrophs was redesigned in order to take into account new full-length sequences of this gene, and the PCR primers were validated using DNA templates from a range of methylotrophs and methanotrophs. The marine methyl bromide-utilising bacterium Leisingera methylohalidivorans MB2 had not yet been shown to possess the cmu pathway of methyl halide-utilisation and this was attempted using Southern hybridisation analysis. This study has demonstrated the diversity of cmuA sequences and the potential for organisms possessing these genes to form an important part of the oceanic sink of atmospheric methyl bromide. It has also laid firm foundations for further investigation of these bacteria through the development of GC ECD and molecular microbiological techniques.
1
Chapter 1
Introduction
2
1 Ozone depletion and the methyl halides
1.1 Atmospheric methyl halides
Methyl halides (CH3X, X= Br or Cl) are reactive volatile organic compounds
composed of a halogen atom covalently bonded to the methyl group CH3. Methyl
bromide (CH3Br) and methyl chloride (CH3Cl) are gases under standard conditions,
and methyl iodide is a volatile liquid. All three compounds exhibit varying degrees of
toxicity, but CH3Br in particular has been used to good effect as a fumigant in
agriculture in order to control insect, nematode and other pests in a wide range of
economically important crops. It has found use for pre-plant soil fumigation, post-
harvest protection and in quarantine procedures (Ragsdale & Vick, 2001; Yagi et al.,
1995) and its efficacy for these purposes was confirmed as long ago as 1949 (Goodey,
1949).
CH3Br and CH3Cl are present in the atmosphere as trace gases at levels of ~10 and
600 pptv (parts per trillion by volume), respectively (Khalil & Rasmussen, 1999;
Khalil et al., 1993). Although only present in trace amounts, these CH3Xs have a
large effect on atmospheric chemistry, CH3Cl contributes 15 % to the overall
atmospheric burden of chlorine and CH3Br is the largest carrier of bromine to the
stratosphere (Butler, 2000). Once in the stratosphere, reactions with hydroxyl radicals
and photolysis release the reactive halogen species Br and Cl allowing them to
catalyse in cyclic reactions with ozone (O3), which ultimately result in the destruction
of this compound (Yung et al., 1980). It has also been demonstrated that they
contribute to destruction of tropospheric ozone (Platt & Honninger, 2003). Bromine
is 50-60 times more effective at ozone destruction than chlorine on a per atom basis
3
(Butler, 2000) and as such has been assigned a high Ozone-Depleting Potential of
0.65 (Mellouki et al., 1992). As such, in 1992 under the Copenhagen amendment,
CH3Br has come under the auspices of the ‘Montreal Protocol on Substances that
Deplete the Ozone Layer’, which aims to freeze emissions of ozone-depleting gases at
their 1995 levels for developed countries
(http://www.undp.org/seed/eap/montreal/montreal.htm).
1.2 Sources and sinks
Quantification of sources and sinks of ozone-depleting gases allows the assessment of
how effective bans on usage of these compounds are. With entirely anthropogenically
produced chlorofluorocarbons such as CFC-11 it is possible to monitor their
atmospheric decline in response to the protocol (Walker et al., 2000). With CH3Br
the case is complicated by the fact that there are natural sources and sinks of the
compound as well as anthropogenic production from biomass burning (Andreae &
Merlet, 2001; Mano & Andreae, 1994), car exhausts (Baker et al., 1998), as well as
soil fumigants (Yates et al., 1998). Natural sources of CH3Br include the oceans,
where it is produced by macro-algae (Laturnus, 1995; Laturnus et al., 1998) and
phytoplankton (Saemundsdottir & Matrai, 1998; Scarratt & Moore, 1998); and
terrestrial sources include higher plants (Saini et al., 1995, Rhew et al., 2003;
Yokouchi et al., 2002), fungi (Field et al., 1995; Manley, 2002), coastal salt marshes,
rice paddies and wetlands (Rhew et al., 2000), (Redeker et al., 2002), and (Varner et
al., 1999), degradation of organic matter (Keppler et al., 2000) and even
photochemically induced production within surface snow (Swanson et al., 2002).
Sinks of CH3Br include tropospheric hydroxylation, chemical and biological
degradation in soils (Yates et al., 2003) and also the oceans, which are both a source
4
and sink of CH3Br (Butler, 1994; Tokarczyk et al., 2003), but overall seem to be a net
sink (Groszko & Moore, 1998; Lobert et al., 1995). Atmospheric chemistry requires
that the total amount of CH3Br produced equals the total amount degraded, minus the
standing stocks of CH3Br. Currently atmospheric budgets for CH3Br are unbalanced,
in (Ennis, 1998) sources for only 60 % of the sinks could be accounted for, the
missing sinks totalling 83 Gg/yr of CH3Br. Fig 1.1 displays current estimates of
CH3Br fluxes (Rob Rhew, pers. comm.).
5
Fig 1.1. Global CH3Br budget. Values are the amount of CH3Br in Gg/yr. Yellow arrows indicate sources of CH3Br and blue arrows indicate sinks. There are missing sources which contribute 83 Gg/yr . These are known to be terrestrial and believed to be plant based (based on a figure by Rob Rhew, pers. comm.)
Oceanic production
56 41
83
Biomass burning
CH3Br fumigation
Leaded petrol
Missing source
2 Gg/yr 5 Gg/yr
77
42
86 •OH hv
Soils
Oceans
Atmosphere
6
The global oceans are an interesting case as they are both a source and a sink of
CH3Br (Anbar et al., 1996). CH3Br can be broken down chemically in seawater by
hydrolysis and nucleophilic substitution with Cl- (Elliott & Rowland, 1995; Gentile et
al., 1989). There have been conflicting reports concerning the oceans as a sink for
CH3Br since they were previously thought to be supersaturated with respect to
atmosphere and therefore a strong source of CH3Br (Khalil et al., 1993; Singh &
Kanakidou, 1993). However, more recent work has shown that most oceans are
undersaturated in CH3Br with respect to the atmosphere (Lobert et al., 1997; Lobert et
al., 1995; Tokarczyk & Saltzman, 2001; Tokarczyk et al., 2003). An exception is the
North Atlantic during algal blooms (Baker et al., 1999). Coastal waters are also often
supersaturated (Baker et al., 1999), with one study demonstrating that the sea was
saturated with CH3Br for over three months of the year and that greatest saturation
occurred in conjunction with a bloom of the phytoplankton Phaeocystis (Baker et al.,
1999). Upwelling regions are near equilibrium with respect to atmosphere (Lobert et
al., 1997). The ocean is now thought to be a global net sink of atmospheric CH3Br of
–21 Gg/yr (Lobert et al., 1997).
(King & Saltzman, 1997) determined chemical and biological loss rates for CH3Br
surface ocean waters and demonstrated that biological loss rates were significant in
comparison to chemical loss rates and that biological pathways existed for the
removal of CH3Br from these waters. Both autoclaved and 0.2 µm filtered seawater
samples failed to demonstrate the high rates of CH3Br degradation seen in unamended
samples. Examination of the CH3Br loss rates associated with individual size
fractions of the marine biomass resulted in the discovery that loss of CH3Br was
associated with that fraction that encompassed the bacterial size range. #
7
The oceanic lifetime and fate of CH3Br is an important parameter in global models
seeking to predict the atmospheric response to proposed changes in emissions of
CH3Br. A clearer understanding of the biological mechanisms for the removal of
CH3Br in the marine environment by microorganisms is therefore essential.
1.3 Methylotrophic bacteria
Methylotrophic bacteria are capable of gaining all their carbon and energy needs from
one-carbon compounds more reduced than CO2. Substrates utilised by these
organisms include methane, methanol, methylated amines (e.g. mono-, di-, and
trimethylamine), methylated sulphur species and methyl halides (Murrell &
McDonald, 2000). Both aerobic and anaerobic prokaryotes can make use of
methylotrophic substrates for growth, and aerobic methylotrophs include both Gram
positive and Gram-negative bacteria (Anthony, 1982).
1.3.1 Aerobic methylotrophy
Within the aerobic methylotrophs, a functional distinction is made between obligate
methylotrophs and facultative methylotrophs, which can also use non-methylotrophic
compounds as growth substrates. Obligate methylotrophs can be subdivided into
those that can use methane as sole source of carbon and energy (methanotrophs) and
those that cannot. The CH3X-utilising bacteria that have been isolated so far are all
capable of growth on non-methylotrophic substrates and are hence classified as
facultative methylotrophs. Aerobic methylotrophs play an important part in global
carbon cycling as methane is the most abundant organic gas in the atmosphere
(DeLong, 2000; Murrell & McDonald, 2000).
8
1.3.2 Methanotrophy
Methanotrophs make use of methane monooxygenase enzymes in order to oxidise
methane to methanol, which is further oxidised by methanol dehydrogenase. There
are two forms of this enzyme, a particulate form and a soluble form. Methanotrophs
have been isolated with either one or both of these enzymes, but possession of the
soluble form is a less common trait (Colby et al., 1977; Dedysh et al., 2000). The
methanotrophs are divided into two groups by arrangement of intracellular
membranes, Type I methanotrophs possess bundles of vesicular discs and Type II
methanotrophs have membranes running around the periphery of the cell. The two
types differ from one another in their biochemistry as Type I methanotrophs and other
α-Proteobacterial methylotrophs assimilate carbon at the level of formaldehyde
through the serine pathway, whereas Type II methanotrophs (γ-Proteobacteria)
together with obligate methanol-utilizers of the β-Proteobacteria use the ribulose
monophosphate cycle (RuMP) (Chistoserdova & Lidstrom, 2002).
1.3.3 Biochemistry: carbon assimilation and energy generation
Methylotrophs face a number of metabolic challenges, including building all their
cellular carbon from one carbon compounds and channelling all their assimilatory and
dissimilatory carbon through the toxic intermediate formaldehyde. Certain key
enzymes are involved in this process, including the methane monooxygenases and
methanol dehydrogenase (MDH), both of which have been used as functional
molecular markers of methylotrophy in the environment (Inagaki et al., 2004;
McDonald & Murrell, 1997). Methyl groups of methylotrophic substrates are
oxidised to formaldehyde by various substrate specific oxidases or dehydrogenases.
Formaldehyde can then either be completely oxidised to CO2, or be assimilated into
cellular biomass by the serine or RuMP cycles. If present, the ribulose bisphosphate
9
cycle (Calvin-Benson-Bassham cycle) can also be used to assimilate cellular carbon
from CO2. A summary of aerobic methylotrophy pathways can be seen for a variety
of substrates in Fig 1.2 below.
10
Fig. 1.2. Metabolism of C1 compounds by aerobic methylotrophic bacteria adapted from (Lidstrom, 2001). Numbering refers to the enzymes for each step: 1, methane monooxygenase; 2, methanol dehydrogenase; 3, formaldehyde dehydrogenase; 4, formate dehydrogenase; 5, dichloromethane dehalogenases; 6, methyl halide methyltransferase/corrinoid binding protein; 7, methanesulfonic acid monooxygenase; 8, methylated sulphur dehydrogenases or oxidases; 9, methylated amine dehydrogenases; 10, methylamine oxidase.
11
At least four pathways have been identified for the oxidation of formaldehyde, one
being cyclic and three linear. The cyclic pathway involves a dissimilatory RuMP
cycle and 6-phosphogluconate dehydrogenase (Anthony, 1982), and the first of the
linear pathways is a common mechanism involving a NAD+ linked formaldehyde
dehydrogenase (Duine, 1993). The further two pathways were identified in
Methylobacterium extorquens AM1 where formaldehyde reacts with either
tetrahydromethanopterin, a co-factor previously thought to be specific to
methanogens, or tetrahydrofolate forming the methylene form of the co-factors. The
methylene group is progressively oxidised to CO2 by a series of different enzymes
specific for the particular carrier (Chistoserdova & Lidstrom, 2002).
1.3.4 Anaerobic methylotrophy
Two bacterial processes are involved in anaerobic methylotrophy: methanogenesis
and anaerobic methane oxidation. Methanogens are Archaea that either
disproportionate acetate, formate and selected other C1 compounds to CO2 and CH4,
or reduce them to CH4 using H2. Methanogens reduce the methyl group carrier
methyl coenzyme M (McoM) to methane and oxidised coenzyme M using the enzyme
methyl coenzyme M reductase. Reduction of the coenzyme M is coupled to the
phosphorylation of ADP to ATP, and thus energy production. Methyl groups can be
transferred to McoM via a variety of systems, including tetrahydromethanopterin.
The employment of tetrahydromethanopterin could indicate a link between
methanogenesis and aerobic methylotrophy (Ferry, 1999; Sauer et al., 1997). When
using methanol or methylamine as an energy source specialised methyltransferases
and corrinoid-binding proteins transfer the methyl group directly from these
compounds to coenzyme M (Deppenmeier, 2002). These two enzymes share some
homology with the CmuA methyltransferase/corrinoid binding protein of aerobic
12
methyl halide utilisation (see below), which is similarly reminiscent of the strictly
anaerobic process of methanogenesis and aerobic methylotrophy.
1.4 Bacterial degradation of halogenated compounds
A wide range of different halogenated compounds, including aliphatic and aromatic
halogenated hydrocarbons, exist in the environment and can be degraded aerobically
and anerobically by bacteria using a variety of means. Many of these have significant
anthropogenic sources and investigation into their bacterial degradation is often from
a bioremediation standpoint (Fetzner, 1998). Five strategies for dehalogenation were
defined by (Fetzner, 1998) and include:
• Oxidative dehalogenation as a result of mono- or di- oxygenase-catalysed co-
metabolic reactions (such as co-metabolic degradation of CH3Br by methane
monooxygenase (Dalton & Stirling, 1982).
• Dehydrohydrohalogenase-catalysed dehalogenation involves the removal of
the halogen atom and formation of a double bond. This has been
demonstrated to occur in the degradation of the insecticide lindane by
Sphingomonas paucimobilis UT26 (Fetzner, 1998).
• Substitutive dehalogenation involves either hydrolysis catalysed by
halidohydrolases, or a thiolytic mechanism with glutathione as co-substrate.
Substitutive dehalogenation mechanisms are the most common mechanisms of
dehalogenation and have been found in a wide range of bacteria and acting
upon aliphatic, heterocyclic and aromatic halogenated compounds (Fetzner,
1998).
• Reductive dehalogenation and dehalorespiration. Certain anaerobic bacteria
can use halogenated compounds such as chlorophenols and chloroethenes as
13
electron acceptors. This process is important in the biodegradation of
halogenated pollutants such as trichloroethene in anaerobic environments
(Gerritse et al., 1999).
• Halogen methyltransferases can operate in both anaerobic and aerobic
organisms. The anaerobic homoacetogen Acetobacterium dehalogens uses
CH3Cl as sole energy source, producing acetate (Fetzner, 1998). The aerobic
methyltransferase system is best exemplified by the CH3X-utilisers that are the
subject of this thesis.
1.5 Aerobic utilisation of methyl halides and characteristics of isolates
The first CH3X-utiliser was Hyphomicrobium sp. MC1, isolated by Leisinger and
colleagues in 1986, which was shown to degrade CH3Cl aerobically (Hartmans et al.,
1986). At the time this reaction was suggested to occur via a monooxygenase
reaction rather than a dehalogenation. The strain has subsequently been lost and so
this cannot be confirmed.
1.5.1 Methylobacterium chloromethanicum CM4
Doronina et al., (1996) isolated eight strains of CH3Cl-degrading bacteria from
industrially-contaminated Russian soils. After 16S rRNA phylogenetic analysis it
was revealed that in fact two distinct strains had been isolated, which were designated
as Methylobacterium chloromethanicum CM4 and Hyphomicrobium
chloromethanicum CM2 (McDonald et al., 2001).
The mechanism of CH3Cl metabolism in M. chloromethanicum CM4 was investigated
and two major polypeptides, of 67 kDa and 35 kDa were discovered to be induced
during growth on CH3Cl. Cells grown on CH3Cl were also shown to be capable of
14
oxidising CH3Br and CH3I (Vannelli et al., 1998). Transposon mutagenesis was used
in order to identify the genes responsible for CH3Cl degradation and mutants that
would still grow on other methylotrophic compounds. This information was coupled
with physiological, biochemical and genetic evidence, and a pathway was suggested
for the process (Vannelli et al., 1999) see Fig 1.3).
Fig 1.3. Pathway of CH3Cl degradation in Methylobacterium chloromethanicum CM4 as (Vannelli et al., 1999). Numbering refers to enzyme for that particular step in the pathway: 1. CmuA, methyltransferase/corrinoid protein; 2. CmuB, methyltransferase; 3. MetF, 5,10-methylene-tetrahydrofolate reductase; 4. and 5. FolD, 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (4./5.); 6. PurU, 10-formyl-tetrahydrofolate hydrolase; 7. FDH, Formate dehydrogenase.
Carbon assimilation via serine cycle
CH3Cl
H4folate
CH H4folate
CHO H4folate
CH2 H4folate
CH3 H4folate
HCOOH
CO2
H2O
H2O
H4folate
2 H+
2 H+
2 H+
HCl
CoI
CH3 CoIII
1
4
5
6
3
7
2
15
The first step in this pathway is the transferral of the methyl group of CH3Cl to the
Cobalt atom of a corrinoid group. A single 67 kDa enzyme, CmuA carries out this
task, being a methyltransferase coupled to a corrinoid-binding protein. The second
step involves the methyltransferase CmuB (35 kDa), which transfers the methyl group
from the corrinoid group to tetrahydrofolate forming methyl tetrahydrofolate. Both
these methyltransferases were demonstrated to be essential for growth on CH3Cl by
transposon mutagenesis (Studer et al., 2002). Methyl tetrahydrofolate is then
progressively oxidised to formate and CO2, with carbon assimilation via the serine
cycle at the level of methylene tetrahydrofolate. CmuA and CmuB were purified and
were able to catalyse the transfer of the methyl group of CH3Cl to tetrahydrofolate in
vitro (Studer et al., 2002).
Sequence analysis of the genes involved indicated that they were present on two
clusters on the M. chloromethanicum CM4 genome (see Fig 1.4, later). CmuC and
FolD were also shown to be essential for growth on CH3Cl, but the function of the
methyltransferase CmuC is not yet known.
Vitamin B12 was shown to be required for the growth of M. chloromethanicum on
CH3Cl and that it was not required as an addition to the growth medium as it was
synthesised by the cells. Presence of cobalt in the growth medium was an obligate
requirement (Studer et al., 2002).
1.5.2 Hyphomicrobium sp.
Hyphomicrobium chloromethanicum CM2 was isolated alongside M.
chloromethanicum CM4 (Doronina et al., 1996) and along with this strain it is one of
the most studied CH3X-utilising bacteria. A CH3Cl-inducible enzyme system was
16
shown to be present in H. chloromethanicum CM2 with a 67 kDa CmuA and 35 kDa
CmuB being expressed during growth on CH3Br and CH3Cl (McAnulla et al., 2001b).
A single gene cluster containing the cmuA, cmuB, cmuC and folD genes known to be
essential for growth on CH3Cl was cloned and sequenced. CmuA from H.
chloromethanicum CM4 displayed the same methyltransferase/corrinoid binding
protein structure as with M. chloromethanicum CM4 and shared 80 % identity at the
amino acid level (McDonald et al., 2002). A number of other ORFs were present on
this cluster, including paaE encoding a putative reductase and hutI encoding an
imidazolonepropionase (see Fig 1.4).
Transposon and site-directed mutagenesis studies were carried out in H.
chloromethanicum CM2 (Borodina et al., 2004) and mutational inactivation of cmuB,
cmuC or hutI was shown to negate the ability to use CH3Cl as sole carbon and energy
source. Furthermore it was demonstrated by reverse transcription PCR analysis that
the cmuB, cmuC, cmuA, fmdB, paaE, hutI and metF genes were co-expressed and co-
regulated as parts of a single mRNA transcript. Expression of the transcript was
CH3Cl inducible and not repressed by the presence of the alternative growth substrate
methanol. CH3Cl- transposon mutants were also unable to use CH3Br as sole carbon
energy source, indicating that the same pathway was being used to utilise both
CH3Xs. These analyses indicated that a similar pathway for CH3Cl-degradation
existed in both these Methylobacterium and Hyphomicrobium isolates.
A number of other Hyphomicrobium spp. have been isolated that are capable of
growth on CH3Cl and CH3Br. (McAnulla et al., 2001a) enriched for and isolated six
Hyphomicrobium strains: S-3 was isolated from the Severn estuary, S-4 from
17
Warwick soil, MAR-1 from the North Sea, SAC-1 and SAN-1 and PMC from the
same woodland site. Strains S-3, S-4 and MAR-1 were phylogenetically very similar
to H. chloromethanicum CM2, SAC-1 and SAN-1 in comparative 16S rRNA
sequences analysis grouped together close to, but separate from strain CM2. PMC
was more distantly related. (Borodina et al., 2005) isolated seven further
hyphomicrobia from a range of soils enriched with CH3Cl.
The number of Hyphomicrobium spp. that have been isolated on CH3Xs may indicate
that these bacteria form a significant sink for CH3Cl and CH3Br in soils, although this
may also be due to enrichment conditions favouring these organisms to the detriment
of environmentally more significant organisms. It is not known how active they are
in the environmental degradation of CH3Xs. Borodina et al., (2005) also applied
CH3Cl DNA Stable Isotope Probing (Radajewski et al., 2000) to the same soil
samples and revealed a greater diversity of CH3Cl-utilising bacteria to be present as
demonstrated by analyses of 16S rRNA and functional gene (cmuA) amplification and
sequencing than that from the enrichment cultures.
1.5.3 Aminobacter sp.
Two Aminobacter sp. that are capable of growth on both CH3Cl and CH3Br have been
isolated and have had their cmu (chloromethane-utilising) gene clusters characterised.
They have recently been designated Aminobacter ciceronei IMB1 and Aminobacter
lissarensis CC495 (McDonald et al., In Press). A. ciceronei IMB1 was isolated from
soil that had previously been fumigated with CH3Br (Connell-Hancock et al., 1998;
Miller et al., 1997) and A. lissarensis CC495 was isolated from pristine woodland soil
(Coulter et al., 1999). A. ciceronei IMB1 was able to utilise CH3Cl, CH3Br, and CH3I
as sole carbon and energy sources and CH3Cl and CH3I demonstrated competitive
18
inhibition with CH3Br, suggesting that a common enzyme system was responsible for
utilisation of all three CH3Xs (Schaefer & Oremland, 1999). Growth on all three
CH3Xs was also demonstrated to be inducible and cells grown on CH3Br were
capable of oxidising CH3Cl and vice versa. In addition, this organism was
demonstrated to be able to oxidise tropospheric concentrations (pptv) of CH3Br,
indicating the potential of these bacteria to be important degraders of CH3Br in the
environment (Goodwin et al., 2001). A cmu cluster has been cloned and sequenced
from this organism (Woodall et al., 2001) and contained cmuC, cmuA, paaE, hutI and
metF genes, cmuB was not identified in this strain (see Fig. 1.4). Again, these results
combine to suggest that a similar pathway is in operation in A. ciceronei IMB1.
A. lissarensis CC495 was only able to grow on CH3Cl or CH3Br as sole carbon and
energy source when vitamin B12 was supplied in the medium (Coulter et al., 1999),
in contrast to M. chloromethanicum CM4, H. chloromethanicum CM2, and A.
ciceronei IMB1 (Studer et al., 1999). Growth on CH3Cl and CH3Br was
demonstrated to be inducible and 68 kDa and 28 kDa proteins were shown to be
expressed when cells were grown on CH3Cl , but not when grown with methylamine.
The 68 kDa protein was purified and identified as a halomethane:bisulfide/halide ion
methyltransferase (Coulter et al., 1999).
N-terminal sequences of this protein displayed identity to the derived N-terminus of
CmuA from M. chloromethanicum CM4 (81.3 %), H. chloromethanicum CM2 (68.8
%), and A. ciceronei IMB1 (81.3 %) (McDonald et al., 2002). Southern hybridisation
analysis using a radiolabelled probe based on the cmuA sequence of A. ciceronei
IMB1 allowed the cloning and sequencing of a cmu cluster from A. lissarensis CC495
19
(Warner, 2003; Warner et al., In Press). Similar to H. chloromethanicum CM2 and A.
ciceronei IMB1 genes were arranged in single cluster: cmuB, cmuC, cmuA, paaE and
hutI and shared high identities with the corresponding genes from A. ciceronei IMB1.
It is likely that a pathway similar to that elucidated by (Vannelli et al., 1999) is also in
operation in this case, rather than the halomethane:bisulfide/halide ion methyl
transferase mechanism suggested by Coulter et al., (1999).
1.5.4 Marine CH3Br utilising bacteria
Prior to the start of this investigation, despite the fact marine systems have been
shown to possess significant capacity for biological CH3Br degradation, only two
marine bacteria capable of utilising CH3Br as sole source of carbon and energy had
been isolated: Leisingera methylohalidivorans MB2 (Goodwin et al., 2001; Schaefer
et al., 2002) and strain LIS 3 (Hoeft et al., 2000). L. methylohalidivorans MB2 was
isolated from a CH3Br-degrading seawater enrichment that had been sourced from a
tide-pool of the coast of California (Goodwin et al., 1998). It was able to utilise
CH3Br, CH3Cl, and CH3I as sole sources of carbon and energy and this trait was
demonstrated to be inducible (Schaefer et al., 2002). Alongside A. ciceronei IMB1,
L. methylohalidivorans MB2 was also able to oxidise tropospheric levels of CH3Br.
Attempts have been made to identify a cmuA gene in L. methylohalidivorans MB2,
but have yet to meet with success (Warner, 2003). Southern hybridisation analyses
with probes generated from M. chloromethanicum CM4, H. chloromethanicum CM2,
A. ciceronei IMB1 and A. lissarensis CC495 failed to hybridise with
L. methylohalidivorans MB2 DNA. SDS PAGE analysis of cells grown on complex
marine broth with and without CH3Br failed to reveal any differences in expressed
proteins. The pathway of CH3Br-utilisation remains unknown in this organism.
20
Strain LIS 3 was isolated from a CH3Br and dimethyl sulfide enrichment of Long
Island Sound seawater and inhibition experiments using trichloroethene as an
inhibitor of methyltransferase reactions indicated that it was capable of utilising
CH3Br via a methyltransferase mechanism (Hoeft et al., 2000). This isolate has yet to
be characterised further and so the details of this mechanism also remain unknown.
During the course of this investigation (Schaefer et al., 2005) enriched Plymouth and
Scottish coastal seawater with CH3Br and isolated 13 strains of marine CH3Br-
utilising bacteria belonging to three distinct clades of the α-Proteobacteria. Of these
clades, two were found to make use of the cmu pathway and representatives of each of
these (strains 179 and 198) had their cmu clusters successfully cloned and sequenced.
Sequence analysis indicated the presence of cmuA, fmdB, paaE, hutI and metF in the
fragment of strain 179 cmu cluster, and of cmuC, cmuA, fmdB, paaE, and hutI in
strain 198. A metF sequence could not be identified in strain 198. SDS PAGE
analysis of CH3Br grown cells of strains 179 and 198 compared with glycine betaine
grown cells demonstrated inducible expression of 67 kDa proteins and their identity
was confirmed by mass spectrometry analysis. This indicated that the cmu pathway
of CH3X degradation was present, not only in terrestrial environments, but also in
marine environments. The conservation of structure of all the cmu clusters sequenced
to date can be seen in Fig 1.4.
Strains of the third clade of marine CH3Br-utilising isolates, represented by
Rhodobacteraceae strain 217 (sharing most phylogenetic identity with Roseovarius
strains) did not demonstrate the presence of a 67 kDa protein that was inducibly
expressed when grown on CH3Br. Those proteins that did demonstrate differential
21
expression between CH3Br grown and glycine betaine grown cells could not be
conclusively identified by mass spectrometry. As with L. methylohalidivorans MB2,
it is not known by what mechanism CH3Br is utilised in these strains.
22
Fig 1.4. Comparison of cmu gene clusters sequenced to date. Genes involved in the metabolism of CH3X are in blue with cmuA in red. Genes not directly involved are in green. Organisms are referred to by their strain names.
str. IMB-1
2 kb
I
I
I
str. CC495
str. CM4
str. 198
cmuB cmuC cmuA fmdB paaE hutI
cobU cobD metF cmuB cmuC cobC
cobU folC folD purU cmuA
cmuC cmuA fmdB paaE hutI metF
cmuA fmdB paaE hutI cmuC nrdF nrdA
str. CM2 cmuB cmuC cmuA fmdB paaE hutI metF
//
str. 179 cmuA fmdB paaE hutI metF
23
1.5.5 Phylogeny of CH3X-utilising bacteria
All CH3X-utilising bacteria isolated and characterised so far have been members of
the α-Proteobacteria. A single Gram positive bacterium, which was determined to be
most similar to the Nocardiodes was isolated, but unfortunately the strain was lost
(McAnulla, 2000). Despite the fact they are all within the α-Proteobacteria, the
CH3X-utilising strains are distributed throughout this clade (see Fig 1.5). CH3X-
utilisation is a monophyletic trait in that it has thus far only been found within the α-
Proteobacteria, although within this clade it is sporadically distributed a fact that is
best demonstrated by A. ciceronei strains IMB1, ER2 and C147 (McDonald et al., In
Press). These three strains are all members of the same species, but only A. ciceronei
IMB1 is capable of utilising CH3X as sole carbon and energy sources, strains ER2 and
C147 cannot grow on these compounds and have not been discovered to possess a
cmuA gene.
24
Fig 1.5. Maximum likelihood tree of near full-length 16S rRNA sequences of a selection of the isolated strains of CH3X utilisers, indicating their distribution throughout the α-Proteobacteria. Terrestrial isolates are indicated in red, with marine isolates highlighted in blue. The 16S rRNA sequence of Erythrobacter longus was used to root the tree
25
1.6 Molecular ecology
Traditional microbiological techniques such as the enrichment and isolation of novel
organisms are exceedingly useful tools for determining the ecology of particular
bacterial species. However, the limitation of these approaches is that many organisms
are recalcitrant to culture by traditional means. New culturing approaches, such as
dilution to extinction culturing methods and use of extremely dilute media
demonstrate some promise for accessing microbial biodiversity missed by classical
culturing techniques (Rappe et al., 2002), but molecular ecology techniques can
access much more of this diversity.
Molecular ecology makes use of analyses of individual bacteria and, more commonly,
populations of bacteria at the molecular level in order to gain information about the
function and/or phylogenetic affiliation of that organism or population in the
environment. A wide range of techniques are available and some of the most
common, and powerful techniques are discussed below.
1.6.1 Functional and phylogenetic genetic markers
DNA or RNA samples can be extracted from entire communities of environmental
bacteria. Amplification of genes of functional or phylogenetic interest using the
polymerase chain reaction and specific primers can allow determination of whether a
particular functional gene is present in a given sample, and the diversity of the
organisms bearing those genes can be assessed. The 16S rRNA gene is often targeted
in this way in order to determine the diversity of microbial assemblages, as it is
conserved between all bacteria. Universal bacterial primers have been designed for
the 16S rRNA gene, which amplify various regions. Ligation of these amplimers into
26
plasmid vectors and transformation into bacterial hosts creates a library of sequences
representative of those in the environment. These can be dereplicated by techniques
such as restriction fragment length polymorphism analysis (RFLP) (Borodina et al.,
2005) for example) and divided into operational taxonomic units (OTUs).
Representative clones of OTU can be sequenced and phylogenetic analyses applied in
order to establish the identity and relatedness of the cloned genes to one another.
With the CH3X-utilising bacteria phylogenetic analyses are hampered by the fact that
CH3X degradation is not a monophyletic trait. In this and similar cases functional
genetic markers can be used in order to assess the diversity of bacteria that are
capable of carrying out a particular process associated with the targeted gene in the
environment. Examples of genes that have been used as functional genetic markers
include the pmoA and mmoX genes, which encode the catalytic subunits methane
monooxygenases and have been used as markers of methanotrophy, and mxaF, which
encodes the large subunit of methanol dehydrogenase and has been used as a
functional marker of methylotrophy (Inagaki et al, 2004; McDonald and Murrell
1997).
With CH3X-utilising bacteria cmuA, the gene encoding the first step in the
methyltransferase pathway, has been developed and applied as a functional genetic
marker in a variety of terrestrial environments (Borodina et al., 2005; McAnulla,
2000; Miller et al., 2004; Warner, 2003).
27
1.6.2 Microbial assemblage finger-printing techniques
Clone library analysis can be time-consuming and the cost of sequencing large
numbers of clones in order to be statistically rigorous in representation of the
microbial assemblage can be prohibitive. These factors limit the number of clones
that can be analysed and may result in libraries that severely underestimate the
diversity present in the environmental sample. Techniques, such as denaturing
gradient gel electrophoresis (DGGE, Diez et al., 2001; Freitag & Prosser, 2003) and
terminal restriction fragment length polymorphism (TRFLP, Engebretson & Moyer,
2003; Moeseneder et al., 1999; Osborn et al., 2000) have been developed that allow
the more rapid assessment of the diversity of organisms present in microbial
assemblages. Both techniques are again based on PCR amplification of DNA using
primers specific for particular phylogenetic or functional genetic markers; however,
the primers have been modified, in the case of DGGE by addition of a GC-rich
sequence, and in the case of TRFLP by addition of a 5’ fluorophore.
DGGE PCR products are run on acrylamide gels that contain an increasing
concentration of denaturant from the top to the bottom of the gel. PCR products
denature at positions in the gel that correspond to their sequence, but are held together
by the more strongly bonded GC-clamp. Different sequences halt their
electrophoretic movement at different positions in the gel, thus, after staining and
visualisation of the gels, producing a characteristic banding pattern representative of
the sequence diversity amplified from the particular DNA sample. The intensity of
individual bands can be used as an indicator of the relative dominance of each
sequence type in the sample and excision and sequencing of bands can allow their
identification.
28
TRFLP PCR products are digested with carefully selected restriction enzymes which
are chosen for their ability to discriminate between different sequence variations of
the same gene. These digested products are resolved by DNA sequencing. Only the
terminal restriction fragment corresponding to the 5’ end of the product can be
visualised as it is the only fragment that remains attached to the fluorophore. The size
of these terminal restriction fragments (TRFs) produces the microbial assemblage
fingerprint. The relative fluorescence of each TRF can be used as an indicator of the
relative abundance of particular sequences in a similar manner to intensity of bands in
DGGE analysis, although identification of a particular TRF relies on comparison of
their sizes with known sequences, as TRFs cannot be sequenced.
1.6.3 Localisation of specific microorganisms within environments and
microbial assemblages
The molecular ecological techniques discussed so far allow the identity and diversity
of genes to be assessed in a bulk sample, but do not allow visualisation or
identification of individual organisms within an environment. Fluorescence in situ
hybridisation causes individual cells that possess a specific DNA sequence to
fluoresce and they can then be visualised using fluorescent microscopy (Davenport et
al., 2000). DNA probes are designed for specific sequences of 16S rRNA and
labelled with fluorophores. These are then hybridised with microbial assemblages
fixed to slides and permeablised in order to allow entry of the fluorescent probe to the
cell, where it hybridises to the cognate sequence of 16S rRNA. By using a variety of
probes that hybridise with various taxonomic groups together with a variety of
fluorophores the diversity within a microbial assemblage can be resolved at the level
of individual organisms.
29
One of the drawbacks of FISH has been that it has only been possible to design probes
for ribosomal RNA as these are expressed at high copy numbers in bacteria. Recently
a mRNA FISH technique has been developed that allows simultaneous hybridisation
of probes to mRNA and rRNA and therefore the detection of functional gene
expression alongside determination of phylogenetic identity at the level of individual
cells (Pernthaler & Amann, 2004).
1.6.4 Linking microbial assemblage identity with function
Determining the identity of organisms within an assemblage may give clues to
environmental function they perform, but cannot elucidate this further. Similarly
examination of functional diversity is useful, but can only give clues to the identity of
the organisms performing the function in the environment if the functional trait is
monophyletic. Recently there have been a number of significant developments of
techniques that can link the functional diversity of a microbial assemblage with the
phylogenetic identity of the organisms involved. mRNA FISH is an example of one
of these techniques. Other techniques include the use of micro-autoradiography of
organisms that have been fed with radiolabelled substrates and subsequent FISH
analysis, identifying the organisms in the sample that have been utilising the substrate
(MAR-FISH, Lee et al., 1999). DNA Stable Isotope Probing (SIP, Radajewski et al.,
2000) and the complementary RNA SIP (Manefield et al., 2002) again make use of
labelled substrates, in this case, heavy isotopes such as 13C, which are pulse fed to the
subject assemblage. The heavy isotope is incorporated into the biomass of that
portion of the microbial assemblage that is actively utilising the substrate in question.
DNA or RNA from the total population can be extracted and separated by density
30
gradient centrifugation, as nucleic acids from the active portion of the assemblage are
heavier than that of the non-active portion. These nucleic acids can then be subjected
to the other molecular techniques mentioned in order to identify the active population,
thus linking population identity with environmental function.
1.7 Sampling sites
Two main sampling sites were used during this investigation, L4, a sampling station
off the coast of Plymouth which could be visited up to weekly, and the Arabian Sea as
part of the AMBITION (Analysing Microbial Biodiversity In The Indian OceaN)
NERC thematic cruise for the Marine and Freshwater Microbial Biodiversity thematic
programme.
1.7.1 Station L4
Station L4 has been visited weekly by scientists based at Plymouth Marine Laboratory
and the Marine Biological Association of the UK since 1988 (see Fig 1.6). Research
vessels RVS Squilla, RVS Sepia and RVS Plymouth Quest bring back weekly water
samples from this station for laboratory analysis and conduct in situ depth profiles of
CTD (conductivity, temperature and depth) and chlorophyll a abundance. Datasets
for zooplankton identification and abundance from this sampling site extend back to
1988 and from 1992 physical, chemical and biological measurements have been taken
including phytoplankton identity and abundance. The datasets are freely available
from the L4 website www.pml.ac.uk/L4/.
31
Fig 1.6. Location of sampling station L4 in the English Channel. L4 is located 10 nautical miles South of Plymouth and is subject to weak seasonal stratification. Phytoplankton composition is characterised by spring diatom and summer dinoflagellate blooms (information from www.pml.ac.uk/L4/Location.htm).
1.7.2 Arabian Sea
The Arabian Sea has been used as a sampling site by a large number of investigators,
including the global programs JGOFS (Joint Global Ocean Flux Study) and WOCE
(World Ocean Circulation Experiment). The Arabian Sea and Indian Ocean are often
selected for analysis because, despite being one of the smallest ocean basins, they
contain a diversity of biogeochemical provinces including eutrophic, oligotrophic,
upwelling and oxygen deplete environments (Burkhill et al., 1993). In 2001 from the
30th August to 29th September, RRS Charles Darwin research cruise CD132
completed a transect (see Fig. 1.7.) of the Arabian Sea in order to characterise the
32
microbial diversity present. 11 stations were sampled along the 5 500 km transect,
mainly following the 67o E meridian. The cruise was collaborative with participants
studying a range of different bacteria and making a wide range of complimentary
measurements including nutrients, photosynthetic pigments, phytoplankton abundance
and production. Microorganisms studied included the Bacteroidetes, pico-eukaryotes,
and nitrogen-fixing bacteria. Data are available from the BODC (Biological
Oceanography Data Centre) and the cruise report contains full details of
measurements taken. Some data are included in Appendix A, and a list of samples
taken is included in Appendix B.
33
Fig 1.7. AMBITION cruise track. The track is marked in red, with sampling stations indicated in yellow.
34
1.8 Aims
Given the importance of marine systems in CH3Br cycling, and the potentially
significant role of bacteria as a sink of CH3X in the marine environment the aims of
this project were four-fold:
• Measurement of environmental (pptv) concentrations of CH3Br in seawater
• Enrichment, isolation, and characterisation of CH3Br-utilising bacteria
• Use of molecular ecological analyses to determine the presence, distribution
and diversity of CH3Br-utilising bacteria in seawater
• Correlation of the presence and concentration of CH3Br with the presence and
abundance of CH3Br-utilising bacteria in seawater
The studentship was funded by NERC and tied to a larger thematic program, Marine
and Freshwater Microbial Biodiversity. Funding was also provided on the project for
a PDRA, Dr. Hendrik Schäfer. The principal and co-investigators were Prof. Colin
Murrell and Dr. Ian McDonald at the University of Warwick and Dr. Phil Nightingale
at Plymouth Marine Laboratory.
35
Chapter 2
Materials and Methods
36
2 Chapter 2: Materials and Methods
2.1 Bacterial and Algal Strains
The bacterial and algal strains used in this study are given in Table 2.1.
Strain Characteristics Source Reference Methylobacterium chloromethanicum strain CM4
MeX utiliser Prof. Yuri Trotsenko
(Doronina et al., 1996)
Hyphomicrobium chloromethanicum strain CM2
MeX utiliser Prof. Yuri Trotsenko
(Doronina et al., 1996)
Aminobacter strain IMB-1 MeX utiliser Dr. Larry Miller
(Connell-Hancock et al., 1998)
Aminobacter strain CC495 MeX utiliser Prof. David Harper
(Coulter et al., 1999)
Leisingera methylohalidivorans strain MB2
MeX utiliser Dr. Kelly Goodwin
(Goodwin et al., 1998)
Ruegeria strain 198 MeX utiliser Dr. Hendrik Schäfer
(Schaefer et al, 2004)
Strain 179 MeX utiliser Dr. Hendrik Schäfer
(Schaefer et al, 2004)
Roseovarius strain 217 MeX utiliser Dr. Hendrik Schäfer
(Schaefer et al, 2004)
Silicibacter pomeroyi strain DSS3
Related to marine MeX utilisers
Dr. J Gonzalez
(Gonzalez et al., 2003)
Strain DSS8 Related to marine MeX utilisers
Dr. J Gonzalez
(Gonzalez et al., 2003)
Roseobacter agricola Related to marine MeX utilisers
Dr. J Gonzalez
(Gonzalez et al., 2003)
Methylobacterium extorquens AM1
Possesses a Methanol dehydrogenase
Warwick
Methylococcus capsulatus (Bath)
Possesses a Methanol dehydrogenase
Warwick (Whittenbury et al., 1970)
Methylophilus methylotrophus W3A1
Possesses a Methanol dehydrogenase
Prof. Nigel Scrutton
Flavobacterium strain RD4.3
Methylotroph without Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Mycobacterium ratisbonense strain EM3
Methylotroph without Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Pseudomonas strain PM2 Methanol utiliser Dr. Paolo de Marco
(Pacheco et al., 2003)
37
Strain Characteristics Source Reference Escherichia coli strain TOP10
Cloning host strain Invitrogen Corporation
TOPO TA cloning Kit
Emiliania huxleyi 92A MeX Producer Dr. Declan Schroeder
Emiliania huxleyi 373 MeX Producer Dr. Declan Schroeder
Emiliania huxleyi 373 UEA
MeX Producer Dr. Declan Schroeder
Emiliania huxleyi 379 MeX Producer Dr. Declan Schroeder
Emiliania huxleyi 1516 MeX Producer Dr. Declan Schroeder
Emiliania huxleyi 1516 CCMP
MeX Producer Dr. Declan Schroeder
Table 2.1. Bacterial and algal strains used; available references are indicated.
2.2 Genomic DNA Samples and Plasmids
Genomic DNA samples used in this study are recorded in Table 2.2.
Strain Characteristics Source Reference Afipia felis strain 25Ei Possesses a methanol
dehydrogenase Dr. Azra al-Moosvi
(Moosvi et al., 2005)
Marinosulfonomonas strain TR3
Possesses a Methanol dehydrogenase
Warwick
Methylosinus trichosporium OB3b
Possesses a Methanol dehydrogenase
Warwick
Methylosinus sporium 5 Possesses a Methanol dehydrogenase
Warwick
Methylocystis parvus Possesses a Methanol dehydrogenase
Warwick
Methylomonas methanica S1
Possesses a Methanol dehydrogenase
Warwick
Methylomonas rubra Possesses a Methanol dehydrogenase
Warwick
Methylomicrobium album BG8
Possesses a Methanol dehydrogenase
Warwick
Methylomonas agile A20 Possesses a Methanol dehydrogenase
Warwick
Hyphomicrobium strain P2 Possesses a Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Methylobacterium strain P3
Possesses a Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Ancylobacter strain SC5.10
Possesses a Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
38
Strain Characteristics Source Reference Methylobacterium strain PM1
Possesses a Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Methylophilus strain ECd4 Possesses a Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Ralstonia strain EHg5 Methylotroph with no Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Rhodococcus strain RD6.2 Methylotroph without Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
Arthrobacter strain SK1.18
Methylotroph without Methanol dehydrogenase
Dr. Paolo de Marco
(De Marco et al., 2004)
TOPO Vector Cloning Plasmid Invitrogen Corporation
TOPO TA cloning Kit
Table 2.2. Genomic DNA extracts used; available references are included.
2.3 Media
Liquid media were prepared as described below. The corresponding agars were
prepared by the addition of 1.5 % (w/v) Bacto agar (Difco) to the respective liquid
media prior to autoclaving. All media were autoclaved at 121 oC for 15 min.
39
2.3.1 MAMS (Marine Ammonium Mineral Salts)
MAMS medium was used for growth of Leisingera methylohalidivorans strain MB2
and also for enrichments and was adapted from Thompson et al., 1995; the SL-10
trace elements solution as described by Widdel et al., 1983.
Amount (per L)
deionised water 970.0 mL
NaCl 20.0 g
(NH4)2SO4 (200 g/L stock soln.) 5.0 mL
100 x CaCl2 stock soln. (2.0 g/100 mL) 10.0 mL
100 x MS soln. 10.0 mL
SL-10 Trace Elements soln. 1.0 mL
Methyl Halide Vitamin soln. 5.0 mL
100 x Phosphate Stock soln. 10.0 mL
Adjust pH to 7.0-7.3 if required, and add phosphates and vitamins solutions
aseptically after autoclaving for 15 min at 121 oC.
100 x MS Solution
Amount (per 100 mL)
MgSO4.7H2O 10 g
FeSO4.7H2O 0.02 g
Na2WO4 (0.1 mM in 20 mM NaOH) 1.0 mL
Na2MO4.2H2O (0.2 g/mL solution) 1.0 mL
Made using deionised water and autoclaved
40
100 x Phosphates Stock Solution
Amount (g/100 mL)
KH2PO4 3.6
K2HPO4 23.4
Made using Milli Q water and autoclaved
Methyl Halide Vitamin Solution
Amount (mg/L)
Thiamine HCl 10
Nicotinic acid 20
Pyridoxine HCl 20
p-Aminobenzoic acid 200
Riboflavin 20
Biotin 1
Cyanocobalamine (Vit. B12) 200
Folic acid 4
Filter sterilized and stored refrigerated.
MAMSTY (Marine Ammonium Mineral Salts with Tryptone and Yeast Extract)
This is a complex medium based on MAMS with the addition of 1.0 g/L yeast extract
and 5.0 g/L tryptone prior to autoclaving.
2.3.2 10 x ANMS (Ammonium Nitrate Mineral Salts) Medium
This was made as described by Whittenbury et al., 1970 as a base for the marine
methylotroph growth medium described below. The other stock components, except
the vitamin solution described below can also be found in (Whittenbury et al., 1970).
41
2.3.3 Marine Methylotroph Growth Medium
Amount (per L)
10 x ANMS 10.0 mL
ANMS Trace Elements 1.0 mL
ANMS Molybdate Stock 0.5 mL
ANMS Fe-EDTA Stock 0.1 mL
ANMS Phosphate Stock 10.0 mL
Methyl Halide Vitamin Solution 5.0 mL
NaCl 35.0 g
After autoclaving, add phosphates and vitamins aseptically. ANMS is used at 0.1 x
above strength.
2.3.4 Choi Medium
This was used for the growth of Methylobacterium extorquens strain AM1 as
described by Bourque et al., 1995 except that 5.37 g/L of Na2HPO4.12H2O was used
in place of 4.02 g/L of Na2HPO4.7H2O. Filter sterilized methanol (Aristar , BDH
Laboratory Supplies) at 0.5 % (v/v) was used as the carbon source.
2.3.5 NMS (Nitrate Mineral Salts)
Medium for growth of Methylococcus capsulatus (Bath) was made as described by
Whittenbury et al., 1970 with 10 % (vol/vol) methane in the headspace as carbon
source.
2.3.6 C2 Medium
This was used for growth of Methylophilus methylotrophus W3A1 as described by
Colby & Zatman, 1973. A stock solution of 10 % (w/v) TMA (trimethylamine) was
filter sterilized and added to a final concentration of 0.3 % (w/v).
42
2.3.7 Luria-Bertani Medium
This was routinely used for the growth of Escherichia coli and prepared as described
by Sambrook & Russell, 2001.
2.3.8 f/2-Si Medium
This medium was used for the culture of all Emiliania huxleyi strains. The recipe
used was that of the Provasoli-Guillard National Centre for Culture of Marine
Phytoplankton (CCMP) and can be found on their website at the URL
http://ccmp.bigelow.org/CI/f2_family.html (Guillard & Ryther, 1962; Guillard, 1975).
It was made using aged and filter-sterilised and aged seawater from the English
Channel.
2.4 Growth and maintenance of bacterial cultures
All strains except E.coli were routinely grown in 25 mL of media in 125-mL serum
vials. Those that were grown on a MeX substrate were closed with blue Teflon
coated butyl rubber stoppers and crimp sealed. All cultures were grown with orbital
shaking at 200 rpm.
2.5 Microscopy
A Zeiss Axioskop (Germany) microscope with phase contrast and oil immersion was
routinely used to examine bacterial cultures and enrichments.
2.6 Sample collection and storage
Seawater samples were collected using a variety of different techniques depending on
the source of the sample and the availability of equipment at the time of sampling.
43
2.6.1 Arabian Sea samples
Water samples were taken using a SeaBird rosette sampler equipped within 24 x 30-L
Niskin bottles and CTD (conductivity, temperature and depth) devices. The exact
configuration of the system can be found in the AMBITION Cruise report available
from the Biological Oceanographic Data Centre website at the following URL:
www.bodc.ac.uk/projects/m&fmb.html. The Niskin bottles were sub-sampled using
their integral taps and a short length of Tygon tubing into 2 L polycarbonate bottles
rinsed three times with seawater sample. Water was treated in one of two ways. It
was filtered through 47 mm Polyethersulfone Supor-200 0.2 µm filters (Pall-Gellman)
using Nalgene filter housings and transferred to cryovials. These were subsequently
flash frozen in liquid nitrogen and stored at –80 oC. Alternatively water was filtered
through 0.2 µm Sterivex filter cartridges, which were sealed at either end with
Nescofilm, flash frozen and stored in 15 mL Falcon tubes at –80 oC.
At the end of the Cruise, the samples were packed in copious quantities of dry ice in
polystyrene boxes for the transfer from Oman to the UK. After no more than four
days in dry ice, they were transferred back to –80 oC where they remained until DNA
was extracted. Dry ice remained in the boxes when they were opened, indicating the
temperature had been maintained. A list of samples can be found in Appendix B.
2.6.2 Station L4 Samples
L4 Samples were collected using individual 5 L Niskin bottles and transferred to 2 L
polycarbonate screw-cap bottles. Surface water samples were collected in the same
type of bottles using the non-toxic seawater supply pump of the vessels RVS Squilla
and RVS Sepia. When larger volumes of surface water samples were required, 20 L
44
carboys (Nalgene) were filled from the non-toxic supply and sub-sampled back at the
laboratory.
2.6.3 Celtic Sea Samples
These samples were provided by Dr. Gary Smerdon of Plymouth Marine Laboratory
and were taken during a cruise aboard RRS Discovery (D261) in the Celtic Sea from
the 1st to 14th of April 2002.
2.7 Enrichment and isolation of CH3X-utilising marine bacteria
During the AMBITION Cruise alongside the filtering of water for DNA extraction
2 L of water were filtered through 47 mm, 0.2 µm Supor filters and the filtrate was
then resuspended in ~3 mL of sample water. This was repeated for both the 5 m
depth sample and the chlorophyll maximum sample, the depth of which was
determined by the CTD profile at each station. At station 6 an extra set of samples
were taken from the deep cast of 2501 m. An extra set at was also taken at 250 m at
station 8, together with a final extra set at station 11 at the salinity maximum. See
Appendix B for a complete list of samples.
100 µL of the filtrate suspension was added to each of twelve pre-prepared 25 ml
enrichment vials containing 5 ml of 0.1 x ANMS with 3.5 % (w/v) NaCl, ANMS trace
elements and the following 200 x vitamin solution, used at 1 x final concentration.
Amount (mg/L)
Folic Acid 4
p-aminobenzoic acid 200
Cyanocobalamine 200
45
Seven different carbon sources were used, either individually, or in combination with
one another, and at various concentrations, resulting in a set of twelve enrichments
being prepared for each sample.
2.7.1 Enrichment Conditions
Twelve different enrichment conditions were used on the cruise with the carbon
sources as shown in table 2.3.
Vial Carbon Source Gas Concentrations 1 0.1 % (vol/vol) CH3Br 85.9 µM CH3Br 2 0.5 % (vol/vol) CH3Br 429.3 µM CH3Br 3 1.0 % (vol/vol) CH3Br 859.7 µM CH3Br 4 50 mM Methanol 5 50 mM Methanol and
0.5 % (vol/vol) CH3Br 429.3 µM CH3Br
6 10 mM Methylamine 7 10 mM Methylamine and
0.5 % (vol/vol) CH3Br 429.3 µM CH3Br
8 10 mM Formate 9 10 mM Formate and
0.5 % (vol/vol) CH3Br 429.3 µM CH3Br
10 10 % (vol/vol) Methane 145.0 µM Methane 11 2 % (vol/vol) CH3Cl l 1536.5 µM CH3Cl 12 10 mM L-Methionine and
0.5 % (vol/vol) CH3Br 429.3 µM CH3Br
Table 2.3. Cruise enrichment conditions. Gas concentrations are calculated as described in Appendix C using the Henry’s law constants of De Bruyn & Saltzman, 1997.
2.7.2 A Note on gas concentrations in media
For the sake of practicality, gaseous carbon sources were added to the pre-sealed
crimp-top vials as a percentage of the headspace volume of the vial. Henry’s Law
was then used in order to calculate the concentration of the substrate in the aqueous
phase. The concentrations found with the most commonly used enrichment volume
formats for CH3Br and CH3Cl can be found in Table 2.4, below.
46
Gas Temp. (oC)
[Headspace] (%)
Headspace Volume (mL)
Medium Volume (mL)
[Medium] (µM)
CH3Br 20 0.2 100 25 176.9 CH3Br 30 0.2 100 25 148.6 CH3Br 20 0.2 300 700 128.0 CH3Br 20 0.1 19 5 85.9 CH3Cl 20 2 100 25 1574.8 CH3Cl 30 2 100 25 1173.8 CH3Cl 20 2 300 700 1176.8 CH3Cl 20 2 19 5 1536.5 Table 2.4. Selected concentrations of CH3X depending on the culture format employed. Headspace concentrations of 0.2 % (v/v) and 2 % (v/v) for CH3Br and CH3Cl
respectively were found to be the highest that did not exhibit toxicity as determined
by restriction of growth in enrichment culture. Enrichments could not be maintained
on higher concentrations of these CH3X.
2.8 O2 Electrode
A Rank Brothers (Cambridge, UK) digital Clark-type Model 10 oxygen electrode
connected to a Churchill thermocirculator was used for potentiometric studies. A
2 mL electrode chamber was used and the change in potential (oxygen consumption)
recorded on a Philips PM8251A one-line chart recorder. Sodium dithionate crystals
and air saturated de-ionised water were used to calibrate the electrode. The method
followed was that of Thompson et al., 1995. Gaseous substrates and substrates or
inhibitors with only limited water solubility were added as µl volumes of saturated
solutions prepared in stoppered and crimp-sealed 125 ml glass vials.
2.9 Gas Chromatography
Three gas chromatographic (GC) systems were used during the project, a GC with
flame ionisation detection (GC FID) and two GCs with electron capture detection
(GC ECD). The GC FID system was based at the University of Warwick in the
47
Department of Biological Sciences, and the GC ECD in the Biogas and Tracer Group
of Plymouth Marine Laboratory.
2.9.1 GC FID
This GC system (‘system one’) was used to determine the presence or absence of
CH3X in the headspace of cultures and enrichments. 100 µl of headspace gas was
injected manually into a GCD Gas chromatograph (PYE Unicam Ltd., Cambridge,
UK) fitted with a 1 m x 4 mm glass column containing Poropak Q (Phase Separations
Ltd., Deeside, UK). Oxygen-free N2 was used as the carrier gas at a flow rate of 30
mL min-1 and the oven temperature was 200 oC. The flame ionisation detector
generated peaks in potential and these were integrated by a 3390A Integrator (Hewlett
Packard, Berkshire, UK). The gas chromatograph was calibrated with known
amounts of standards using a range of dilutions of CH3Br and CH3Cl. Typical
retention times were 1.10 min and 1.65 min for CH3Cl and CH3Br respectively.
2.9.2 GC-Electron Capture Detection
The GC-ECD system (‘system two’) based at PML was initially designed around a
Shimadzu 8A gas chromatograph with custom built air purifiers and purge and trap
apparatus. The carrier gas was ECD grade He, with N2 as make-up and sparge gases.
After identification of an electronic problem with this system, the GC was changed to
a system previously used for the detection of fluorocarbons “system three” (Haine et
al., 1995). Both systems were partially automated. For further details of the design
and specifics of these systems see Chapter 3.
48
2.10 General Purpose Buffers and Solutions
TE buffer, 10 x TBE buffer, Southern hybridisation buffers and 6 x agarose gel-
loading buffer (with Ficoll and Bromophenol Blue) were prepared and used as
described in Sambrook & Russell, 2001.
2.11 DNA extraction methods
2.11.1 Hot phenol extraction
This method was used for the preparation of nucleic acids from environmental
samples concentrated on 0.2 µm Supor filters. The polyethersulfone from which these
filters are made is phenol-soluble, thus all the cells from the filter are released during
this procedure. The method used was that of Schaefer & Muyzer, 2001; briefly filters
are rinsed with ice-cold buffer and then cells are lysed by the addition of SDS and hot
(65 oC) phenol followed by incubation at 65 oC and frequent vortex mixing.
Subsequent phenol:chloroform:isoamyl alocohol (25:24:1) extractions result in DNA
purification and DNA is precipitated using sodium acetate. DNA was resuspended in
50 µl or 100 µl of sterile deionised water, by mixing overnight at 0-5 oC and then split
into two equal aliquots. A working stock was kept at –20 oC and a reserve stock was
stored at -80 oC.
2.11.2 DNA extraction from Sterivex filters
As the Sterivex filters are completely enclosed inside a cartridge casing, DNA
extraction was carried out using the method of Somerville et al., 1989. SDS,
lysozyme and proteinase K incubations accomplish cell lysis before subsequent
phenol:chloroform extractions and DNA precipitation. This method generally yielded
lower amounts of DNA in comparison to the hot phenol method as determined by
49
agarose gel electrophoresis, presumably due to adherence of the filtered biomass to
the surface of the filter.
2.12 Gel Electrophoresis
Depending upon the separation required 1-2 % agarose gels were prepared and run in
1 x TBE buffer. Small gels were run using a Flowgen minigel systems (Flowgen
Instruments Ltd., Sittingbourne, UK). Larger gels for Southern hybridisation were
run on BRL model H4 horizontal gel systems (Bethesda Research Laboratories,
Cambridge, UK). RFLP analysis was performed on 2 % agarose gels on the same
systems. For minigels, 0.5 µg/ml ethidium bromide was included during the casting
of the gels; for the larger gels, staining was carried out after electrophoresis by
soaking in 1 x TBE with 0.5 µg/mL ethidium bromide with gentle orbital shaking for
60 min. Destaining was carried out for 30 min in 1 x TBE. DNA was visualised by
placing the stained gels on a UV transilluminator and photographed using an instant
camera (CU5 Land Camera) loaded with Polaroid 665 black and white film.
Exposure times varied according to the intensity of the products. Alternatively,
particularly for the larger gels, a Gel Documentation system was used.
2.13 Quantification of DNA
This was carried out by two methods, depending upon the accuracy of measurement
required. For approximate determination of DNA concentrations, dilutions of DNA
solution were electrophoresed alongside with the Invitrogen 1 kb ladder at a
concentration of 0.5 µg/lane. At this concentration the 1 636 bp band is present at 50
ng. After ethidium bromide staining, the intensity of this band was compared to the
intensity of the DNA to be quantified and an estimate of DNA concentration was then
calculated.
50
For applications when more accuracy was required, or to compare the amount of
DNA produced from different DNA extractions a Nanodrop Spectrophotomer
(Nanodrop) was used to give concentrations of DNA. This system had the advantage
of being able to indicate the purity of the DNA sample, since protein concentration
would also be determined. This service was provided by the University of Warwick
Central Molecular Biology Services Laboratory.
2.14 Gel extraction of DNA
After electrophoresis, gel fragments were excised using an ethanol cleaned scalpel
blade and the DNA extracted using the QIAquick gel extraction kit (Qiagen)
according to the manufacturer’s instructions.
2.15 Restriction digests
Restriction digests of DNA were generally performed in 20 µL volumes in 0.5 mL
Eppendorf tubes with 10 U of enzyme, according to the manufacturer’s instructions.
A range of enzymes from different suppliers was used and these are indicated in the
text. Restriction digests for RFLP analyses were performed in 10 µL reaction
volumes and those for TRFLP analyses were carried out in 100 µL reaction volumes.
See below for details.
2.16 PCR
2.16.1 PCR reaction mixtures and conditions
PCR reaction mixtures were 2.5 mM MgCl2, 200 µM each dNTP, 10-25 pmol of each
primer (dependent on reaction), 1.3 M betaine, 1.3 % (vol/vol) DMSO, in 1 x
Invitrogen Taq DNA Polymerase buffer and 2.5 U of Taq DNA Polymerase
51
(Invitrogen, Paisley, UK) in a total volume of 50 µL, made up with sterile deionised
water. Thermal cycling was carried out on a Hybaid Touchdown thermal cycler with
initial denaturation at 95 oC for 5 min, whereupon the Taq DNA Polymerase was
added as a hot start. This was followed by 35 cycles of 1 min at 95 oC, 1 min at the
primers’ annealing temperature (see table 2.5), and 1 min at 72 oC, followed by final
extension step of 72 oC for 10 min.
2.16.2 PCR Primers
Table 2.5 lists the primers used for both the PCR and in sequencing reactions together
with details of the annealing temperatures used.
52
Primer Sequence (5’-3’)
Annealing temperature
Reference
CmuAF802 TTCAACGGCGAYATGTATCCYGG 55 oC* (Miller et al., 2004)
CmuAR1609 TCTCGATGAACTGCTCRGGCT 55 oC* (Miller et al., 2004)
CmuAF229 CTTTTYACKCCRGTGGAATGCGT 55 oC (Warner, 2003)
CmuAR824 CCRGGATACATRTCGCCGTTGAA N/A* This thesis cmuAR1244 TABTCCATKATBGCYTCGAC 55 oC Dr. Hendrik
Schäfer cmuAF1225 GTCGARGCVATMATGGAVTA 55 oC This thesis cmuAR1352 TCRCCVACGAYYTTCATSCC 55 oC This thesis 27F AGAGTTTGATCMTGGCTCAG 60 oC* (Lane,
1991) 1492R TACGGYTACCTTGTTACGACTT 60 oC* (Lane,
1991) 341F CCTACGGGAGGCAGCAG N/A* (Muyzer et
al., 1993) M13F GTAAAACGACGGCCA N/A* Invitrogen
Corporation M13R CAGGAAACAGCTATGA N/A* Invitrogen
Corporation mxaF1003 GCGGCACCAACTGGGGCTGGT 55 oC (McDonald
& Murrell, 1997)
mxaR1561 GGGCAGCATGAAGGGCTCCC 55 oC (McDonald & Murrell, 1997)
mxaR1555 CATGAABGGCTCCCARTCCAT 55 oC This thesis Table 2.5. PCR Primers used. An * indicates that the primer has been used successfully in sequencing reactions. Primer pairs used in the PCR are as follows: cmuAF802/cmuAR1609, cmuAF229/cmuAR1609, cmuAF1225/cmuAR1352 for cmuA amplification; 27F/1492R for 16S rRNA amplification; MxaF1003/MxaR1561 and MxaF1003/MxaR1555 for mxaF amplification.
2.17 Southern Hybridisation Analysis
Southern blotting (Sambrook & Russell, 2001) was used to transfer DNA onto Nylon
Hybond-N membranes (Amersham, Little Chalfont, UK). DNA was fixed to the
membrane using a UV Stratalinker (Stratagene, Cambridge, UK). Probes were
produced by PCR amplification of the desired segment of the target gene. The DNA
53
fragments were subsequently radiolabelled by the random priming method of
Feinburg & Vogelstein, 1983, with 50 ng of PCR product being labelled with 50 µCi
of 32P dGTP. Probes were denatured by addition of NaOH to a final concentration of
0.4 M prior to use in hybridisation reactions.
Hybridisations were performed according to the method of Feinburg & Vogelstein,
1983 using the buffers of Sambrook & Russell, 2001in a Hybaid oven (Hybaid Ltd.,
Middlesex, UK) with washing stringencies as described by Oakley & Murrell, 1988.
Removal of bound probe from membranes before reuse with other hybridisation
probes was achieved by boiling in 0.1 % (w/v) SDS for at least 10 min.
Fuji nif RX medical X-ray film was used for all microautoradiographs. Radioactive
membranes were exposed to this film in light-tight autoradiography cassettes with two
intensifying screens. Cassettes were stored at –80 oC for between 2 and 96 hr
depending on the signal intensity. Autoradiographs were developed using a Curix 60
automatic X-ray film developer (AGFA).
2.18 Cloning and Clone Library Dereplication
Cloning of PCR products was performed using the TOPO TA cloning kit (Invitrogen)
according to the manufacturer’s instructions. E.coli TOP10 cells were transformed
using the manufacturer’s chemical transformation method and plated according to the
manufacturer’s instructions for blue/white colony screening. Positive (white) clones
were picked onto LB agar containing 50 µg/mL Ampicillin. Master plates of 50
clones each were produced and kept at 0-5 oC for short-term storage. For long-term
storage, clones were grown in 10 mL of LB broth with 50 µg/mL Ampicillin added
from a stock of 100 mg/mL (filter sterilized and stored at –20 oC) and incubated at
54
37oC and 200 rpm shaking in an orbital shaker overnight. The cells were centrifuged
in order to obtain cell pellets, resuspended in 30 % (vol/vol) glycerol and stored at –
80oC.
For dereplication of the clone libraries, all clones were inoculated into 10 mL LB
broth using sterile wooden toothpicks as for the construction of glycerol stocks. After
growth of the overnight culture, 2 mL was taken for use in the alkaline lysis mini-prep
procedure of Sambrook & Russell, 2001. Plasmid DNA then was resuspended in
50 µl of sterile deionised water and was then subjected to restriction fragment length
polymorphism analysis as outlined below.
2.19 Restriction Fragment Length Polymorphism (RFLP) analysis
Restriction digests were performed on plasmid DNAin order to dereplicate the clone
libraries. Digests were carried out in a total volume of 10 µl in 0.5 ml
microcentrifuge tubes, with 2 µl of plasmid DNA and 0.5 U of restriction enzyme.
Double digests of plasmid DNA were with EcoRI, in order to liberate the cloned
insert from the vector, and another enzyme (in the case of cmuA either RsaI or DdeI).
0.25 U of each enzyme was used. Buffers were used according to the manufacturer’s
guidelines and the volume was made up to 10 µl with sterile Milli Q water. 2 µl of
100 µg/mL RNase (Promega) was added to reaction mixes in order to prevent RNA
smears obscuring restriction patterns. Restriction digests were incubated at the
manufacturer’s recommended temperature in a water bath for 16 hours. Loading
buffer was then added to each of the reaction mixtures and the entire volume was
loaded onto agarose mini-gels for electrophoresis. Gels were then stained with
ethidium bromide (EtBr) in order to enable visualisation of the DNA fragments. For
large clone libraries, 500 mL gels cast with 72 wells were used and these gave better
55
resolution of RFLP patterns than the mini-gels. Operational Taxonomic Units
(OTUs) contained within the clone libraries were defined as groups of clones
containing plasmid with unique restriction enzyme patterns. The identity of OTUs
was confirmed by sequencing.
2.20 DNA Sequencing
DNA sequencing was performed in the University of Warwick Central Molecular
Biology Services Laboratory using the BigDye dyedeoxyterminator ready reaction kit
(Applied Biosystems, Warrington, UK) and ABI3100 capillary DNA sequencers.
2.21 Sequence Analysis, Alignment and Phylogenetics
Analysis of 16S rRNA gene sequences was carried out by using the BLAST program
(Altschul et al., 1997) at http://ncbi.nlm.nih.gov/BLAST. The sequences were
aligned with the highest scoring hits using the fast-aligner included with the ARB
software (Ludwig et al., 2004) and the same software was used to produce
phylogenetic trees using the 16S rRNA database provided with the software.
Functional gene sequences were analysed using BLAST to check their identity and
then imported directly into “in-house” ARB databases set up for the relevant gene of
interest.
Phylogenetic trees were calculated using the neighbour-joining, DNAPars (maximum
parsiomony) and AxML (maximum likelihood) programs available in ARB.
Bootstrapping was carried out with at least 100 replicates in neighbour-joining and
DNAPars analyses and the trees produced by each algorithm compared to ensure the
stability of nodes. Bootstrapping for neighbour-joining analysis was carried out in
56
PHYLIP independently of ARB due to a known issue with ARBs implementation of
this algorithm. Trees were rooted with an appropriate relative in each case.
2.22 Terminal Restriction Fragment Length Polymorphism (TRFLP)
analysis
The method used was that of Moeseneder et al., 1999 with certain modifications. The
primers used were cmuAF802 with 5’ 6-carboxyfluoroscein (FAM) labelling and
cmuAR1609 with 5’ 6-carboxy-2’,4,4’,5’7,7’-hexachlorofluoroscein (HEX) labelling
(TAGN, Newcastle). The primers were provided high performance liquid
chromatography (HPLC) purified in order to preclude fluorescent label unbound to
primer and prevent it from interfering with peak detection. Primers were used as for
PCR reactions at 25 pmol per reaction. The PCR reactions were not precipitated prior
to gel extraction as described in the Moeseneder et al., 1999 method, but gels were
cast with wells large enough to take the entire volume of the reaction. This avoided
any potential loss of PCR product during the precipitation. Restriction enzymes
BsiYI (Roche), HaeIII (Helena biosciences) and HpaII (Helena biosciences) were
found to give the best discrimination between OTUs in either the forward, reverse or
both terminal restriction fragment lengths (TRFs); this was determined by analysis of
previously obtained cmuA sequences from cmuA clone libraries of marine
enrichments, using ARB sequence alignments (see also Chapter 6 and Appendix D).
Definition of an OTU was dependent upon the level of analysis. It was defined either
as a single TRF produced by a single restriction enzyme, or as the collation of each of
the three TRFs for each gene sequence. Amounts of product were determined on a
per sample basis by the University of Warwick Central Molecular Biology Services
Laboratory and samples were run with ROX 500 ladder in de-ionised HiDi formamide
on an ABI3100 capillary sequencer running in Genescan mode. Data were analysed
57
using Genemapper v.3.0 (Applied Biosystems, Warrington, UK). Genemapper
analysis parameters are discussed in Chapter 6.
58
Chapter 3
Measurement of Methyl Bromide
59
3 Chapter 3: Measurement of Methyl Bromide
3.1 Introduction
One of the aims of this project was to couple the molecular analysis of CH3X
degrading bacteria with measurements of CH3Br in the same seawater samples.
Gas/liquid chromatography was the natural choice for measurement of CH3Br as
many other investigators have demonstrated (e.g. Nightingale et al., 1995; Cicerone et
al., 1988; Grimsrud & Miller, 1978).
Gas/liquid chromatography (referred to henceforth as gas chromatography) involves
the separation of the component gases of a sample by means of a gaseous mobile
phase along a narrow tube, known as the column, which is coated with a liquid
stationary phase. The components of the sample are retarded at different rates
depending upon their partition into the liquid phase and can be identified by their
retention times on the column. Different compounds require different detectors to
maximise the sensitivity of the system and different column types and compositions to
maximise separation of the compound of interest from the others in the sample.
3.1.1 GC columns
There are many different types of column used in gas chromatography, depending on
the compounds you wish to differentiate, the composition of the sample and the
solvent used. Factors important in the separation of the sample include the length of
the column, the internal diameter of the column, the nature of the liquid phase, the
carrier gas used, and the composition of any support included for the liquid phase.
Two common types of column are discussed here.
60
Packed columns are commonly glass or stainless steel tubes which are packed with a
porous support material such as diatomaceous earth. The packing can be coated with
the liquid phase for the separation. The nature of the column means that they tend to
be shorter and have a wider internal diameter, which can limit the separation.
Capillary columns, with internal diameters of 0.18 to 0.53 mm are made from fused
silica and are coated with a polyamide polymer. This means they can be much longer
as they are more flexible and can be coiled, with lengths of 100 m being possible.
The liquid phase can coat or be chemically bonded to the inside of the column.
Some columns, such as PLOT columns (Porous Layer Open Tubular) do not have a
liquid phase at all and rely on separation between the carrier gas and a solid phase
bonded to the glass of the column.
3.1.2 GC detectors
Once the sample components have been separated from one another they must be
detected. There is a large range of different types of detector and they vary in their
selectivity and sensitivity, which in turn affects their potential applications. A
selection of common detectors and an approximate lower detection limit is shown in
table 3.1.
There were two detector types used in this study, a flame-ionisation detector (FID)
and two electron capture detectors (ECD). The FID is much less sensitive to CH3Br,
but it is simpler to run. It was used to demonstrate the presence or absence of the
relatively high concentrations with respect to atmosphere of CH3Br used in
enrichments and culture of CH3Br utilisers. With the improved sensitivity of the ECD
61
to halogenated compounds it is possible to measure the parts per trillion by volume
(pptv) levels of CH3Br in the atmosphere.
Detector Selectivity Lower Detection Limit ECD (Electron Capture Detector)
Halides, nitrates, nitriles, peroxides, anhydrides, organometallics
50 fg
FID (Flame Ionisation Detector)
Most organic compounds 100 pg
FPD (Flame Photometric Detector)
Sulphur, phosphorous, tin, boron, arsenic, germanium, selenium, and chromium containing compounds
100 pg
PID (Photo-ionisation Detector)
Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics
2 pg
TCD (Thermal Conductivity Detector)
Universal 1 ng
Table 3.1. A selection of common detectors used in gas chromatography. Adapted from technical information available from www.agilent.com.
3.1.3 Theory of electron capture detection
The ECD was invented by James Lovelock in 1959 (Lovelock, 2000; Lovelock,
1963). He used it to make measurements of compounds such as methyl iodide and the
chlorofluorocarbons (CFCs) in the marine boundary layer (Lovelock, 1973). It
enabled measurements of pesticides to pptv levels with such data informing Rachel
Carson’s book The Silent Spring in 1962 and empowering the environmental
movement.
The detector consists of a source of β-particles, normally 63Ni and two electrodes in a
sealed chamber. Make-up gas molecules, such as N2, are supplied constantly and
collide with the β-particles, ionising them and providing a stable electron cloud within
62
the detector (Fig 3.1). A constant current is maintained across this cloud by the
electrodes. As electronegative compounds from the column enter the detector, they
absorb electrons from this cloud and disturb the current. The detector’s electronics
compensate for this, maintaining the current, and the level of the perturbation is
equivalent to the concentration of the compound entering the detector. The fact that
the compound in order for it to be measured must disturb the electron cloud, lends the
detector its selectivity. Halogenated compounds disturb it significantly, whilst
hydrocarbons do not and cannot be detected at all. Oxygen strongly affects the signal
due to its high electronegativity and as such the make-up gas and carrier gas (which is
chemically inert and carries the sample to the detector) must be free of oxygen. It
also reduces the lifetime of the 63Ni β-emitter by oxidising it. Oxygen and water can
also deactivate the column coatings resulting in poor separation of compounds.
Fig 3.1. Diagrammatic representation of the Electron Capture Detector. The cathode is the casing of the detector.
3.1.4 Sample collection
Sample collection is a critical step in the analysis of seawater samples. Turbulence or
mixing with ambient air can alter the amounts of CH3Br present in the sample
especially if the sample is under- or over- saturated with respect to the ambient air.
Samples should be collected in gas tight vessels preferably of brown glass to prevent
63
photolysis. Vessels should also be completely filled, avoiding the presence of both
headspace and bubbles in order to prevent exchange of the dissolved gases with the
gaseous phase.
For the study at L4 sub-samples were taken from Niskin bottles, which can be
winched to the desired depth and fired and sealed remotely, in 300 mL darkened BOD
(Biological Oxygen Demand) bottles without headspace, avoiding bubbles, and
allowing the sample to over-flow before capping and avoiding contamination sources
such as ship’s exhaust.
3.1.5 Analysis of seawater
Samples for gas chromatographic analysis are normally gaseous, or easily volatilised.
Measurements of trace gases in seawater have their own inherent difficulties. Firstly
the levels of CH3Br present can be very low. At L4 the lowest concentration was
0.23 pmol dm-3 (8 % saturation with respect to atmosphere), and thus samples require
concentration prior to analysis. Secondly, the CH3Br needs to be completely stripped
from the seawater sample, as water adversely affects the ECD and seawater is also
corrosive to the mechanical components of the GC system.
Purge and trap methods offer the perfect solution to this (e.g. Krysell & Nightingale,
1994). The apparatus in Fig. 3.2 displays the major components of a purge and trap
system. The seawater sample is passed into the sparge tower from a gas-tight glass
syringe (1, syringe not shown). A purified gas, inert with respect to the compound
you are analysing, is bubbled through the seawater sample for enough time to strip the
dissolved gasses from it (A). Water is removed from this gas stream by a rage of
means that can include chemical driers, such as magnesium perchlorate (4), or by
64
physical means, such as condensation (3) or counter-current exchangers (5). Finally
the gas is passed through a collecting loop that is held above liquid nitrogen (not
shown). The gas of interest freezes and concentrates in the loop. Once the sample
has finished sparging a valve is thrown which enables carrier gas to pass through the
Fig. 3.2. Purge apparatus for GC ECD system two. Numbering refers to items in the text: 1. Sample inlet, 2. Sparge tower, 3. Condensation tube, 4. Magnesium perchlorate drying tube, 5. Nafion counter current exchange drier, 6. Bubble flow meter. Gas flow direction is indicated: A. Sparge gas inlet, B. Sparge gas containing sample outlet, C. Counter-current gas inlet.
loop, which is rapidly heated, driving the concentrated sample onto the column in a
single pulse. Both ECD systems (systems two and three) employed this methodology
for analysis of CH3Br from seawater samples.
3.1.6 Advantages of automation
It was decided that the GC systems used for CH3Br measurement should be
automated as far as possible. This has several advantages, including the fact that it
facilitates operation of the system during ship-based fieldwork. Under any
circumstances it can be difficult to throw the valves in time in the correct sequence
and in a reproducible way, this is rendered more difficult when the system is at sea.
Automation of the system using computer- or integrator-driven programming allows
the reduction or removal of much of the variability between samples
1
2 3
4
5
4
A
B
Fig 3.4. Diagram of GC ECD System two. Solid black lines indicate 1/8 “ Swag
Fig 3.4. Diagram of GC ECD System two. Solid black lines indicate 1/8 “ Swagelok stainless steel tubing, except in the case of the cryofocussing loop which is 1/16 “ Swagelok stainless steel tubing. The area in the dashed box is the water sample purging tower and drying apparatus. C
65
3.2 System one
This system had flame ionisation detection of CH3Br and was also able to detect
CH3Cl. It was fitted with a Porapak Q packed column. It was used to monitor the
disappearance of CH3Br from enrichment cultures and was calibrated by using a range
of standard concentrations of CH3Br. Samples were injected manually through a
septum. See methods section for further details. Owing to the nucleophilic attack by
chloride ions in the media and seawater of enrichments, CH3Br became gradually
substituted to CH3Cl and degradation of the two methyl halides could only be
separated when consumption rates by the enrichment exceeded the substitution rate.
Enrichments were considered to have consumed all the CH3Br when both CH3Br and
CH3Cl peaks were undetectable (Schaefer et al., 2005).
66
3.3 System two
The core of this system was a Shimadzu GC 14A ECD gas chromatograph with an HP
PLOT Q 30 m 0.53 i.d. megabore column and integration by a Chromjet
Spectraphysics integrator. A gas purifier system was built using swagelok connectors
and water and O2 strippers (Supelco, UK) in order to purify the carrier (He), make-up,
sparge and Nafion counter-flow (all N2) gases (see Fig. 3.3)
Fig 3.3. Gas purifier system for removing water, oxygen and other contaminating molecules (such as hydrocarbons) from the carrier, make-up, sparge and Nafion counter flow gases prior to entering the GC system two.
The system was initially designed to be fully automated, with a system of solenoid
and 6-port valco valves for taking in, sparging and removing the seawater sample,
cryotrapping and release of the sample to the GC column and the switching of sample
stream between the main column and a pre-column (see Fig 3.4). All tubing was 1/8”
or 1/16” stainless steel and the valves that came into direct contact with seawater were
Hastalloy C rather than stainless steel as this has greater long-term resistance to the
67
corrosive effects of seawater. The presence of a pre-column in the system allowed the
sample to be diverted once the CH3Br peak had been detected, preventing other
compounds present in the sample from entering the main column and speeding up the
analysis time as there was no need to wait for these other compounds to leave the
main column before starting another sample. Flow rates for the carrier and make-up
gases ranged between 1-5 and 30-40 ml min-1 respectively as the system was being
tested for measurement of CH3Br and the GC oven was run isothermally at 60oC (the
coolest stable temperature setting) with the detector at 320oC.
CH3Br was detectable using the system, but seemed to be extremely variable in the
seawater samples from L4 used to test it. It was unknown at the time whether this
was an artefact of the sample or analysis methodology or a true representation of
natural variability. It was also discovered by using pure CH3Br diluted in laboratory
air as a standard that there seemed to be a fault with the electronics of the GC, which
resulted in the signal output remaining off-scale despite the actual signal being
transient, a fault known as ‘latch-up’. Owing to this problem and attention being
required on other aspects of the project this system was abandoned.
68 Fig 3.4. Diagram of GC ECD System two. Solid black lines indicate 1/8 “ Swagelok stainless steel tubing, except in the case of the cryofocussing loop which is 1/16 “ Swagelok stainless steel tubing. The area in the dashed box is the water sample purging tower and drying apparatus.
69
3.4 System three
Rather than starting from scratch system three was based on a system previously used
for the detection of chlorofluorocarbons (Haine et al., 1995) and the testing and
validation of the system for measurement of CH3Br was started by Malcolm Liddicoat
at PML, continued by myself; the system was finally used to measure natural
concentrations of CH3Br in seawater at L4 by Malcolm. The system was partially
automated with programmable control from a Chromjet Spectraphysics integrator and
actual integration of the signal performed by a personal computer running
ChromPerfect version 3.52 (Justice Innovations Inc., California, US), which
facilitated data handling. The column was a DB-624, 60 m, 0.32 mm widebore
capillary column with 8 µm film from J&W Scientific, which had been found to give
better separation than the HP PLOT Q column used on system two (Malcolm
Liddicoat pers. comm.). The gas purifier set-up was not required for this GC as the
gases were supplied in BIP cylinders (Built-in purifier) by Air Products (UK). The
flow rate of the N2 make-up gas was 30 ml min–1 and the He carrier gas was
5 ml min-1. The sensitivity of the ECD changed whilst the system was in use as the
detector was ageing and the 63Ni source becoming attenuated, by both natural
radioactive decay and the oxidation of the 63Ni. This resulted in the make-up gas
flow-rate having to be increased to 55 ml min-1 in order to ensure the same level of
sensitivity. A gravimetric standard of 500 ppm CH3Br (BOC, UK) was measured
with each batch of runs allowing standardisation despite this.
200 mL seawater samples were sparged for 20 min with BIP N2 at 110-120 ml min-1,
which had been demonstrated to remove CH3Br from the sample to a level below the
detection limits of the system (Malcolm Liddicoat pers. comm.). The gas stream was
70
dried using two magnesium perchlorate drying tubes and cryotrapped on a 1/16”
stainless steel loop held above liquid N2 and maintained at –150oC for the 20 min
sparge time. After 20 min valves were thrown automatically and simultaneously the
liquid nitrogen removed and replaced with boiling water to drive the gas sample onto
the column.
3.5 L4 results
Samples were taken from station L4 for CH3Br measurement from 10th July 2003 to
23rd November 2004, with a hiatus from March to August 2004, as the GC was
required for a cruise. Data were corrected for cryofocussing loop volume (50 µl to
1000 µl), and calibrated against a 500 ppb standard, which corrected for natural drift
of the detector. The detector was changed on the 6th October 2003 and the standard
runs, which were carried out at least once with each batch of samples and usually two
or three times, allowed continuation of the data set. Contaminated samples and those
with no calibration were discarded to produce the graphs in Fig 3.5.
Saturations of CH3Br in the water column seem to vary quite strongly and rapidly and
are reported relative to atmospheric CH3Br concentration (100 % being at
atmospheric concentration, < 100 % undersaturated and > 100 % supersaturated. On
the 13th October 2003 saturations of 164.0 % were measured at 50 m, and became
progressively less saturated until 47.3 % was measured at 10 m depth. The following
week (21st October 2003) levels in the water column were fairly uniform with large
undersaturations of 16.9, 16.3 and 12.9 % measured at 50 m, 10 m and 0 m
respectively.
71
This suggests that CH3Br was being actively produced and rapidly degraded. The
peaks in CH3Br supersaturations occurred in August and September of both 2003 and
2004 and it is a shame that there is gap in the data between March and August 2004 as
this would presumably have demonstrated the transition from undersaturated to
supersaturated levels of CH3Br in the water column.
Comparison of the CH3Br data with the available data from L4 on phytoplankton
abundance (2003 only) indicated that the peak in CH3Br correlated with peaks in the
abundance of both phytoplankton in general and species known to be CH3Br
producers such as Emiliania huxleyi (Fig 3.6). Pearson correlation analysis indicated
that CH3Br abundance was significantly positively correlated with both E. huxleyi and
colourless dinoflagellate abundance. It is interesting to note that the peak in CH3Br
lags behind the peaks in phytoplankton abundance, this agrees with observations that
it is produced by phytoplankton entering stationary phase and senescence/death.
My hypothesis would be that CH3X degrading bacteria bloom in response to the
elevated levels of CH3Br at L4 as the phytoplankton reach their stationary phase and
that the bacteria are responsible for the rapid switch from supersaturated to
undersaturated that can be seen. It is also possible that the CH3Br utilising bacteria
bloom in response to elevated levels of other nutrients released by the lysis of
phytoplankton, and degrade CH3Br alongside these other carbon and energy sources.
The data gives a tantalising glimpse of this possibility, but as there is no molecular or
bacterial evidence that correlates with this data set it is impossible to test this
hypothesis.
72
Enrichments from L4 were inoculated from samples collected on the 18th April 2002,
20th June 2002 and 30th July 2002, and cmuA PCR products detected from all three
enrichments, indicating the presence of bacteria capable of utilising CH3X at all these
times. If patterns of previous years are followed it is likely that the July, and possibly
June samples were taken during a period of supersaturation of CH3Br with respect to
the atmosphere and the April sample during a period of undersaturation.
73
Fig 3.5. L4 CH3Br measurements. Data is expressed as % saturation with respect to atmosphere and the solubility of CH3Br was calculated using the method of (De Bruyn & Saltzman, 1997). The first sample data point was 02/07/2003. The data shown in red are CH3Br measurements irrespective of the depth of sampling. Those in pink are water column means. The gap prior to day 200 of sampling is present as although sampling had begun, data was discarded due to poor calibrations.
74
Fig 3.6. Phytoplankton abundance and CH3Br concentration at L4. ‘Picos’ refers to picoeukaryotes, CH3Br data are the water column means and also averaged when there was more than one reading per week. L4 data from the L4 plankton monitoring programme, Plymouth Marine Laboratory, available from http://www.pml.ac.uk/L4/.
75
3.6 Potential proxies for CH3Br measurement
CH3Br measurements were not taken on the AMBITION cruise, as the GCD ECD
system two which was available at the time was not operational. It is possible to
hypothesise the presence of CH3Br at the sampling stations from other measurements
that were taken.
3.6.1 Pigments
A large number of different macro- (Laturnus, 1995; Laturnus et al., 1998) and micro-
algal (Saemundsdottir & Matrai, 1998; Scarratt & Moore, 1998) species have been
demonstrated to produce CH3Br in laboratory culture (table 3.2).
Certain marine phytoplankton are known to have characteristic pigments which can
be extracted from seawater samples and analysed by HPLC, giving an indication of
the presence of these groups in the samples. Table 3.3 indicates groups of organisms
and the chlorophylls, carotenoids and biliproteins that can be linked with them.
76
Class Organism Culture
collection ID Study
Diatom Chaetoceros diversum (Saemundsdottir & Matrai, 1998)
Chaetoceros atlanticus (Saemundsdottir & Matrai, 1998)
Chaetoceros calcitrans CCMP315 (Scarratt & Moore, 1998)
Dinophyte Amphidinium carterae (Saemundsdottir & Matrai, 1998)
Prorocentrum micans (Saemundsdottir & Matrai, 1998)
Prorocentrum sp. CCMP703 (Scarratt & Moore, 1998)
Prorocentrum tricornatum (Scarratt & Moore, 1998)
Prasinophyte Pynococcus provasolii (Saemundsdottir & Matrai, 1998)
Prymnesiophyte Phaeocystis sp. (Saemundsdottir & Matrai, 1998)
Phaeocystis sp. (Stefels & Van Boekel, 1993)
(Scarratt & Moore, 1998)
Haptophyte Emiliania huxleyi CCMP373 (Scarratt & Moore, 1998)
Cyanobacteria Synechococcus sp. CCMP1334 (Scarratt & Moore, 1998)
Rhodophyta Porphyridium sp. UTEX190 (Scarratt & Moore, 1998)
Table 3.2. Marine phytoplankton demonstrated to produce CH3Br in laboratory cultures. CCMP is the Provasoli-Guillard National Centre for Culture of Marine Phytoplankton, Maine, US. UTEX is the University of Texas Culture Collection of Algae.
77
Pigment Class Pigment (abbreviation) Phytoplankton Group Chlorophylls Chlorophyll a (Chla) All photosynthetic micro-
algae, except prochlorophytes.
Divinyl chlorophyll a (DvChla) Prochlorophytes. Chlorophyll b (Chlb) Green: chlorophytes,
prasinophytes, euglenophytes.
Divinyl chlorophyll b (DvChlb) Prochlorophytes. Carotenoids Peridinin (Per) Dinoflagellates. 19’-Butanoyloxyfucoxanthin (But) Some prymnesiophytes
one chrysophyte, several dinoflagellates.
Fucoxanthin (Fuc) Diatoms, prymnesiophytes, chrysophytes, raphidophytes and several dinoflagellates.
19’-Hexanoyloxyfucoxanthin (Hex) Prymnesiophytes and several dinoflagellates
Violaxanthin (Vio) Green algae: chlorophytes, prasinophytes, eustigmatophytes.
Diadinoxanthin (Ddx) Diatoms, dinoflagellates, prymnesiophytes, chrysophytes, raphidophytes, euglenophytes.
Alloxanthin (Allo) Cryptophytes. Zeaxanthin (Zea) Cyanophytes,
prochlorophytes, rhodophytes, chlorophytes, eustigmatophytes.
Lutein (Lut) Green algae: chlorophytes, prasinophytes.
Table 3.3. Linking pigment presence with classes of phytoplankton (Baker et al., 1999; Schluter & Mohlenberg, 2003; Wright et al., 1991)
78
In terms of using pigments as markers of the presence of classes of phytoplankton
whose members have been demonstrated to produce CH3Br in laboratory culture, the
pigments of interest are fucoxanthin and diadinoxanthin (diatoms, that were actually
dinoflagellates,prymnesiophytes); peridinin (dinoflagellates); 19’-
hexanoyloxyfucoxanthin (dinoflagellates and prymnesiophytes); violaxanthin and
Lutein (prasinophytes); 19’-butanoyloxyfucoxanthin and (prymnesiophytes) of those
measured on the AMBITION cruise (Fig. 3.7).
79
Fig 3.7. Pigment concentrations during the AMBITION cruise in ng L-1. Please note the changing scales of different pigments. Maximum pigment levels were 106.04 ng L-1 Alloxanthin (stn 11), 1013.69 ng L-1 19’-Butanoyloxyfucoxanthin (stn 10), 157.41 ng L-1 Diadinoxanthin (stn 10), 2165.26 ng L-1 Fucoxanthin (stn 9), 376.68 ng L-1 19’-Hexanoyloxyfucoxanthin (stn 8), 3367.1 ng L-1 Chlorophyll a, 14.83 ng L-1 Lutein (stn 10), 279.35 ng L-1 Peridinin (stn 9), and 42.6 ng L-1 Violaxanthin (stn 10). In all cases the maximum pigment levels were at the chlorophyll maximum for that station based on fluorimetry measurements (RRS Charles Darwin 132 Cruise report, ).
80
At every station pigments characteristic of clades whose members have been
demonstrated to produce CH3Br in laboratory culture could be shown to be present,
with increasing concentrations and hence abundance towards the northerly eutrophic
stations. It is possible to hypothesise that CH3Br was therefore produced in these
waters. There are important caveats however; not all members of the phytoplankton
clades shown to produce CH3Br are capable of CH3Br production; it is not known
what the physiological state of the phytoplankton was at the time of sampling as
production has been linked to stationary phase and senescence/degradation; and
pigments are not generally restricted to particular groups that have been shown to be
CH3Br producers (Saemundsdottir & Matrai, 1998; Scarratt & Moore, 1998).
3.6.2 Sea surface temperature (SST)
Sea surface temperature has been correlated with saturation and undersaturation
anomalies of CH3Br (King et al., 2002). The variability in SST has been shown to
contribute to one-half to two-thirds of the variability in methyl bromide oceanic
saturations. The remaining variability is accounted by for other factors including
biological production and degradation. Data from six cruises was fitted to two
quadratic equations for spring and summer, and autumn and winter. The equations
reproduce the saturation anomaly for CH3Br on a global scale, but fail to reproduce
accurately the anomaly as measured on regional scales. At the time of the
AMBITION cruise the equations would predict an undersaturation with respect to
atmosphere of approximately –15 to –20 %. Measurements of CH3Br in
phytoplankton blooms have demonstrated supersaturations (Baker et al., 1999;
Wingenter et al., 2004) and it is likely that this was the case in the eutrophic northern
cruise stations where phytoplankton production was abundant (see Appendix C). A
81
further limitation is that this method only indicates saturation anomaly with respect to
atmosphere. It cannot indicate whether CH3Br is produced and quickly utilised by
bacteria, or whether it is produced at a greater rate than consumption, either of which
could be measured as both super- and under-saturations.
3.7 Discussion
The difficulty with which a GC system was constructed that was capable of
measuring the extremely low CH3Br concentrations in seawater samples left us unable
to precisely correlate the presence and abundance of CH3Br with the molecular
ecological data of the presence and diversity of cmuA and hence the presence of
bacteria capable of using CH3X as sole carbon and energy sources. Methyl bromide
measurements were taken at L4 and demonstrated that levels varied from extremely
undersaturated (lowest value 7.9 % saturation or 0.23 pmol/l, January 2004) to
supersaturated (highest value 214.6 % saturation or 4.70 pmol/l, September 2003) and
that this showed some consistency with season and the abundance of phytoplankton.
The problems with GC system two and its apparent measurement of highly variable
CH3Br levels may have been due to natural variations in the levels of the compound,
as demonstrated by the measurements with system three noted above. It still remains
that there were also electronic problems with this system, which justifies the
abandonment of this system and switch to system three.
Potential proxies for the direct measurement of CH3Br were investigated in order to
gain an appreciation of the levels of the compound during the AMBITION cruise, but
found to be either qualitative (pigment measurements) or not applicable/inaccurate at
the required scale (the models of King et al., 2002).
82
In future now that the system three GC is capable of sensitive and calibrated
measurements of CH3Br it should be facile to take simultaneous samples for the
evaluation of the microbial population capable of degrading the compound. It might
also be possible to take measurements of pigment abundance alongside this in order to
validate the methodology above and estimate the abundance of CH3Br using the
simpler pigment methodology when GC systems are unavailable. It would also be
extremely interesting to investigate how many bacteria capable of utilising CH3Br are
present at L4 throughout the seasonal cycle, using quantitative molecular methods
such as real-time PCR, to see whether this correlates with the observed super-and
under-saturations of the compound in the water column.
83
Chapter 4
Enrichment and Isolation of
CH3Br-utilising bacteria
84
4 Chapter 4: Enrichment and Isolation of CH3Br-Utilising Bacteria
4.1 Introduction
Marine systems are both an important source and sink of CH3Br to and from the
atmosphere (Baker et al., 1999; Tokarczyk et al., 2003). Marine sources of CH3Br
include both micro- and macro-algae (Scarratt & Moore, 1998) and (Laturnus et al.,
1998). Marine sinks are less well understood and believed to be biological and
associated with plankton within the bacterial size range (King & Saltzman, 1997).
When this study was initiated only a single marine strain capable of utilising CH3Br
as sole carbon and energy source had been isolated; Leisingera methylohalidivorans
MB2, isolated from a marine tide-pool in California (see Chapters 1 and 7 for more
information on this strain). No open-ocean strains had been isolated. Hoeft et al.,
(2000) used marine enrichments with combinations of DMS, trimethylamine,
dimethylamine, together with CH3Br as co-substrate in order to isolate DMS and
CH3Br-degrading organisms. They obtained 4 isolates from Long Island Sound one
of which (strain LIS 3) seemed to be able to utilise CH3Br as sole source of carbon
and energy. However they concluded that degradation of CH3Br was a co-metabolic
trait. Further information is unavailable for this strain.
Thermodynamically, using Gibbs free energy values under standard conditions
(Aylward & Findlay, 1986) complete oxidation of CH3Br could provide –723.8 kJ/mol
of energy (Fig 4.1). Using the method of (Heijnen & Van Dijken, 1992), which takes
into account physiological values and a range of electron acceptors, the predicted
growth yield on CH3Br is 17.75 g dry weight/mol (± 30 %).
85
CH3Br + 1.5O2 CO2 + H2O + Br- + H+
-28.0 -16.4 -386.0 -237.2 -104.0 0
Fig. 4.1. Chemical equation for complete oxidation of CH3Br. Standard Gibbs free energy values of formation are included beneath each species.
The aim of this section of work was to enrich for and isolate bacteria capable of
utilising CH3Br as sole carbon and energy source, particularly from the Arabian Sea,
in order to demonstrate the presence of these organisms and to gain insights into their
involvement in marine CH3Br cycling.
86
4.2 Arabian Sea enrichments
During the NERC Thematic Cruise, AMBITION (Cruise CD132, aboard RRS Charles
Darwin), a large number of samples were taken for DNA extraction and molecular
analyses of CH3Br-utilising bacteria. Alongside this an array of enrichments was set
up with a range of carbon sources from all stations in order to enrich any of these
organisms that might be present.
4.2.1 Enrichment production and initial screening
Enrichments were set up in batches of twelve with the carbon sources as listed in
Table 4.1 (see Chapter 2 for more details). At each station 2 L of surface water and
water from the depth of the chlorophyll maximum was filtered and resuspended in 3
mL of water from the same sample. 100 µl of this was added to each vial of the set of
twelve. The enrichments were then stored in a constant temperature room at 20 oC to
await transport to the UK.
Vial Carbon Source 1 0.1 % (vol/vol) MeBr 2 0.5 % (vol/vol) MeBr 3 1.0 % (vol/vol) MeBr 4 50 mM MeOH 5 50 mM MeOH and 0.5 % (vol/vol) MeBr 6 10 mM Methylamine 7 10 mM Methylamine and 0.5 % (vol/vol) MeBr 8 10 mM Formate 9 10 mM Formate and 0.5 % (vol/vol) MeBr 10 10 % (vol/vol) Methane 11 2 % (vol/vol) MeCl 12 10 mM L-Methionine and 0.5 % (vol/vol) MeBr
Table 4.1. Carbon sources in enrichment vials
87
Enrichments were screened after 2 to 8 weeks (depending on whether they were
collected towards the beginning or end of the cruise), by scoring turbidity by eye (as
there was only a small amount of enrichment available it was desirable to retain all of
the sample rather than using a destructive method of biomass measurement, Table
4.2) and by gas chromatography to assess the presence or absence of CH3Br and
CH3Cl.
Station Depth (m)
1 2 3 4 5 6 7 8 9 10 11 12 Vial Number
1 5 12 1* 74 24 2 5 36 2* 62 48 3 5 60 3* 63 72 4 5 84 4* 77 96 5 5 108 5* 36 120 6 2501 132 6 5 144 6* 40 156 7 5 168 7* 49 † 180 8 5 192 8* 29 204 8 250 216 9 2.5 228 9* 7.5 † 240 10 5 † 252 10* 27 † † † † 264 11 5 276 11* 26 288 11 90 300 Table 4.2. Turbidity estimation of Arabian Sea cruise enrichments. White boxes indicate no turbidity; light blue indicates slight turbidity, through to dark blue indicating significant turbidity. The vial number at the end of each row indicates the numbering of the twelfth vial in each set. * Indicates samples considered to be at the chlorophyll maximum by CTD fluorimetry. † Indicates samples which demonstrated CH3X levels below the detection limit of the GC –FID. The two samples that are crossed were damaged during transport back to the UK.
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The vials selected for initial maintenance were those in Table 4.3. CH3Br was added
to the headspace at 0.2 % (vol/vol), which had been found to be the highest level
tolerated without inhibition of growth in other enrichments (Schaefer et al., 2005) and
when growing CH3Br utilising strains. They were then left for a further two weeks, at
which point they were subcultured at 1 % inoculum size into larger 125 ml vials with
20 ml of 0.1X marine ANMS medium (see Chapter 2, Materials and Methods).
Enrichments on CH3Cl were supplied with 2.0 % CH3Cl as this compound is less
toxic and some CH3X-utilising bacteria have demonstrated higher substrate affinities
for this compound, as can be demonstrated by potentiometric (O2 electrode) studies
(see section 4.5).
Vial Number Conditions Station Depth (m) 179 2.0 % CH3Cl 7 49 229 0.1 % CH3Br 9 7.5 243 1.0 % CH3Br 10 5 253 0.1 % CH3Br 10 27 254 0.5 % CH3Br 10 27 255 1.0 % CH3Br 10 27 263 2.0 % CH3Cl 10 27
Table 4.3 Arabian Sea enrichments positive for CH3X utilisation.
The remaining enrichments were split into groups based on whether they displayed
turbidity and on whether they had been exposed to CH3X. Those with CH3X were
monitored for depletion of CH3X in headspace, only four further enrichments were
observed where this was the case, 165, 189, 249 and 273; all four originally enriched
with 10 mM formate and 0.5 % (vol/vol) headspace CH3Br and originating from
stations 7, 8, 10 and 11 respectively at 5 m depth.
Turbid enrichments that had been enriched with substrates other than CH3X were
pooled to form a general methylotrophic pre-enrichment and then given only 0.2 %
CH3Br. The rationale was that providing a less toxic growth substrate initially would
89
allow proliferation of CH3X utilisers that were also capable of growth on other C1
growth substrates and that when supplied with CH3Br they would utilise this more
rapidly than in the case of CH3X only enrichments. 0.1 ml of each turbid enrichment
from conditions 4, 6, 8, and 10 was added to 25 ml of 0.1 x marine ANMS with 0.2 %
(vol/vol) headspace CH3Br in a 125-ml crimp-seal vial. All enrichments were
incubated at room temperature and in the dark to discourage the growth of
phytoplankton.
4.2.2 CH3Br utilisation
The nine subcultured enrichments, together with the pooled enrichment were
examined periodically for utilisation of headspace CH3X. With media containing Cl-
at seawater concentrations (~35 g L-1) CH3Br undergoes nucleophilic substitution to
CH3Cl (Elliott & Rowland, 1993) and therefore enrichments were not considered to
be depleted of CH3X until both compounds were below the detection limit of the GC-
FID. At this point another pulse of the appropriate CH3X was added to the vial after
removing the same volume of headspace in order to keep the pressure inside the vial
constant.
The pooled enrichment stopped oxidising CH3Br after one pulse of 0.2 % (vol/vol)
and was further subcultured to fresh medium at an inoculum size of 5 % in order to
encourage oxidation. This enrichment was labelled PE2 (Pooled Enrichment 2). This
also occurred with the enrichments in table 4.3 above and they were also subcultured.
PE2 actively degraded CH3Br whereas the other subcultured enrichments failed to
oxidise any more CH3Br, either in the subculture or the original enrichment. The
remaining enrichments, all pre-enriched on Formate together with CH3Br continued
oxidising pulses of the compound for varying amounts of time (see table 4.4)
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Enrichment Station Number of 0.2 % CH3Br pulses
Total amount CH3Br consumer (µmoles)
165 7 5 223.2 165.2 7 2 89.2 PE2 Pooled 13 580.4 189 8 2 89.2 249 10 5 223.2 273 11 6 267.9 Table 4.4. Total CH3X consumed by enrichments.
Oxidation of CH3Br by enrichment cultures can be seen in Fig 4.2. The data have
been adjusted against the chemical control (also shown), which indicates the chemical
degradation rate of CH3Br when incubated with autoclaved media, due to nucleophilic
substitution and hydrolysis. A standard curve was unavailable for this data set and so
averages of the CH3Br peak area for all other standard runs of five different
concentrations (1 %, 0.5 %, 0.2 %, 0.05 % and 0.01 %) of CH3Br were taken and a
headspace concentration calculated from the polynomial regression. The R2 value for
this regression was 0.9954.
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Fig 4.2. Oxidation of CH3Br by four enrichments. Rates were calculated for the fastest initial portion of the graph, between 18/10/2002 and 29/10/2002. The chemical loss rate was 0.18 µM CH3Br/day. Oxidation rates were 11.32, 10.03, 8.18 and 4.32 µM CH3Br/day for enrichments 165, 189, 249 and 273 respectively, after adjusting for the chemical control.
4.3 L4 enrichments
The enrichment strategy with L4 samples was different to that of the Arabian Sea
samples. Arabian Sea samples were transferred to media, partly in order to avoid the
complications of setting up vials at sea with a wide range of substrates, two of which
(CH3Br and CH3Cl) were quite toxic. The enrichments were also very small in
volume, to keep transportation problems to a minimum. At L4 large (~1.15 l) crimp-
seal vials were used and filled with 300 ml of surface seawater samples on three
occasions, 18th April (L4.1), 20th June (L4.2), and 30th July (L4.3) 2002. Again they
were incubated at room temperature in the dark and monitored for depletion of 0.2 %
CH3Br by GC-FID. L4.1 consumed 5 pulses of 0.2 % (vol/vol) headspace CH3Br and
L4.2 and L4.3 consumed 3 pulses each, corresponding to 312.5 µmoles and
187.5 µmoles of CH3Br respectively.
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4.4 Non-axenic phytoplankton enrichments
(Schafer et al., 2002) demonstrated that stable relationships existed between uni-algal
diatom cultures and the co-cultured bacteria and that distinct satellite bacteria
assemblages could be found with the algal cultures. Also, axenic and non-axenic
phytoplankton cultures have been demonstrated to produce CH3Br under laboratory
culture conditions (Scarratt & Moore, 1998) and it was hypothesized that it would be
an advantage in non-axenic cultures for the associated bacteria to be able to use
CH3Br as a carbon and energy source. Six cultures of the coccolithophore Emiliania
huxleyi were obtained from the culture collection of Dr. Declan Schroeder (Marine
Biological Association, UK) and flow cytometry was used to confirm whether or not
they were axenic (Table 4.5).
Strain Axenic/Non-Axenic E. huxleyi 92A Non-axenic E. huxleyi 373 Very poor growth, many bacterial sized
particles on flow cytometry E. huxleyi 373 UEA Axenic E. huxleyi 379 Very poor growth, many bacterial sized
particles on flow cytometry E. huxleyi 1516 Non-axenic E. huxleyi 1516 CCMP Non-axenic
Table 4.5. E. huxleyi culture axenicity. Strain designations refer to those of the MBA, UK culture collection.
E. huxleyi strain 1516 CCMP was used as this culture was non-axenic and grew most
readily. Two approaches were used. In approach one, the strain was grown in a 1 L
crimp-seal vial with sterile needles attached to 0.2 µm sterile acrodisc (Pall-Gelman)
filters attached to allow gas exchange. Once the culture had grown the venting
apparatus was removed and CH3Br added to a headspace concentration of 0.2 %
(vol/vol). This was then monitored for depletion of CH3Br. In approach two a
stationary phase culture of E. huxleyi 1516 CCMP was filtered through a 1.2 µm
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47 mm cellulose nitrate membrane filter in order to remove the phytoplankton cells.
20 ml of filtrate containing the satellite bacterial population was added to a 125-ml
crimp-seal vial and given 0.2 % (vol/vol) headspace CH3Br. This vial was also
monitored for depletion of CH3Br. Neither of the enrichments demonstrated loss of
CH3Br over a period of 6 months. This approach has subsequently been successfully
used by Hendrik Schäfer (pers. comm.) to isolate dimethyl sulfide-utilising bacteria,
present as satellite organisms in non-axenic cultures.
4.4.1 Isolation strategy
The isolation strategy used was identical to that of Schaefer et al., 2005 (see also
Chapter 2, Materials and Methods), briefly 50 µl aliquots of Arabian Sea and L4
enrichments were plated in duplicate onto MAMS plates at three different dilutions
(10-2, 10-3 and 10-4) and incubated in anaerobic gas jars (Becton Dickinson) with a
CH3Br atmosphere. Once growth was observed, the colonies on one of each pair of
plates was washed into 5 ml of MAMS and assayed for the utilisation of 0.2 %
(vol/vol) headspace CH3Br. When an assay proved positive for CH3Br utilisation
colonies were picked from the corresponding sister plate, streaked to purity and
maintained for further analysis. Washings from the enrichments 165 at 10-2 and 10-3
dilutions, and the pooled enrichment subculture (PE2) at 10-2 proved positive.
4.4.2 Identification of putative CH3Br-degrading bacteria
15 colonies were picked from the three plates selected above based on differing
colony morphology within each plate. Between plates there was much duplication of
colony morphology with the same morphologies being seen on all three plates.
Strains were streaked to purity over 4-6 weeks and were generally small and slow
growing. Two strains became contaminated with fungi and were lost and MJC 3, 4
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and 13 were extremely slow-growing and resistant to subculture. Direct PCR of the
biomass (see Chapter 2, Materials and Methods) of single colonies of the remaining
strains were used to generate 16S rRNA gene and cmuA PCR products using primer
sets f27/r1492 (Lane, 1991) and cmuAF802/cmuAR1609 respectively. All products
were run on 1 % agarose gels and gel extracted prior to direct sequencing in the case
of 16S rRNA gene PCR products, using primers f27, r1492 (both (Lane, 1991), and
341F (Muyzer et al., 1993), and cloning and sequencing in the case of cmuA PCR
products. See Table 4.6 for results.
A single strain, MJC10 gave a cmuA PCR product and the sequence grouped
phylogenetically with an uncultured marine clade from the Arabian Sea. This strain
had been identified by 16S rRNA sequencing as a Microbacterium sp., of the
suborder Micrococcineae and a Gram positive (confirmed by Gram-staining of the
isolate). MJC10 and the other two isolates identified as Microbacterium were grown
in 125 ml crimp-seal vials on both MAMS and MAMSTY media and supplied with
0.2 % and 0.1 % (vol/vol) CH3Br in the headspace. No utilisation of CH3Br was
observed over a period of 62 days. After DNA extraction, PCR amplification of
cmuA from these cultures proved impossible. It is possible that the MJC10 cmuA
PCR product was contamination, but there are indications that this was not the case.
The sequence of the product was not identical to any of the CH3X-utilising strains, or
any of the sequenced clones. Also, the negative control of the PCR reaction did not
display any contamination. Under these circumstances it could be hypothesized that
the culture was not pure and that a cmuA-possessing culture was present along with
the numerically dominant Microbacterium strain.
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Strain Source Colony morphology 16S rRNA BLASTN result cmuA BLASTN result/ MJC1 PE2 10-2 Flat, white, adherent and
radially lobed. 100 % (1392/1392) AJ244697 Flavobacterium V4.MO.31
Negative
MJC5 PE2 10-2 Small, irregular opaque and yellow.
99 % (1340/1344) Y17227 Microbacterium oxydans
Faint PCR product of expected size
MJC6 165 10-2 ‘Fried-egg’ morphology, translucent with yellow centre
99 % (601/604) AJ244697 Flavobacterium V4.MO.31
Negative
MJC8 165 10-2 Tiny pinprick colonies, reddish brown.
99 % (1333/1336) AJ244716 Erythrobacter-like V4.BO.03
Non-specific amplification (confirmed by sequencing and BLASTN analysis)
MJC10 165 10-2 Small, irregular opaque and yellow.
99 % (1011/1012) Y17237 Microbacterium schleiferi
85 % (601/701) AY934439 Uncultured soil clone. 90 % ID/93 % Sim. AAY46974 Uncultured soil clone.
MJC11 165 10-2 Tiny pinprick colonies, reddish brown.
99 % (1338/1339) AJ244716 Erythrobacter-like V4.BO.03
Negative
MJC12 165 10-3 Flat, white, adherent and radially lobed.
99 % (1349/1355) Y17237 Microbacterium schleiferi
Negative
MJC14 165 10-3 Tiny irregular and white. 99 % (760/764) AJ294340 Erythrobacter citreus HY-6
Negative
MJC15 165 10-3 Tiny irregular and white. 99 % (1340/1341) AJ244716 Erythrobacter-like V4.BO.03
Negative
Table 4.6. Strains isolated from CH3Br enrichments of Arabian Sea samples
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4.5 Oxygen electrode studies of H. chloromethanicum strain CM2
The CH3X-utilising strain Hyphomicrobium chloromethanicum CM2 was used to
examine the substrate affinities and reaction velocity for CH3Cl, CH3Br, and CH3I.
Oxidation rates were calculated for the substrate concentrations given in Table 4.7,
with Km and Vmax values calculated from double reciprocal Lineweaver-Burk plots
in Table 4.8. The data should be viewed only as an indication of relative rates as it is
based on a single replicate. The upper concentrations of substrate used for CH3Br and
CH3I were the highest that did not demonstrate inhibition, either by no oxidation, or a
slowing of the oxidation rate compared with a lower concentration. Substrates were
prepared by using a saturated solution of each of the compounds in sterile deionised
water and concentrations back calculated using the solubility of the compound, and
the smallest volume of these solutions that could accurately be added to the electrode
chamber determined the lower limit.
Substrate Amount of substrate (µmoles) CH3Cl 60, 120, 240, 300, 600, 900 CH3Br 90, 180, 270, 360, 540, 720 CH3I 96, 160, 320
Table 4.7. Amounts of substrate used in O2 electrode studies. It is interesting to note that the order of the upper limits in concentration of the CH3X
reflects the bond strength of the carbon-halide bond.
The mechanism of toxicity of the CH3X is by indiscriminate methylation of cellular
components, and the methylation of DNA by these compounds has been shown to be
the mechanism of carcinogenicity in murine models, with the more weakly bonded
CH3Br and CH3I being more toxic (Bolt & Gansewendt, 1993).
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Substrate Km (µM) Vmax (nmole O2/min/mg dry weight
CH3Cl 80.97 37.58 CH3Br 124.42 45.19 CH3I 300.73 39.05
Table 4.8. Substrate affinity and maximum oxidation rate of H. chloromethanicum CM2 with CH3X.
4.6 Discussion
Of all the enrichments that were set up, from all locations, those from the Arabian Sea
that were enriched initially with formate were the most active. It is interesting to note
that formate dehydrogenase catalyses the final step in the methyl halide degradation
pathway, and perhaps enriching for the presence of this enzyme and the ability to
degrade formate also increased the proportion of the population capable of utilising
CH3Br through the CmuA pathway. However, not all the characterised CH3X
degrading isolates are capable of growth on formate, L. methylohalidivorans MB2 and
Aminobacter ciceronei IMB1 are examples of this, so co- or pre-enrichment with
formate may restrict the diversity of CH3Br-utilising bacteria present in enrichments.
A general trend in turbidity can be observed from Table 4.3, with increased turbidity
in enrichments from more Northerly stations. These waters were more productive
(for example, Station 9 had an all depth average 3H Leucine uptake of 762 pmol/l/h
and Station 1, 47 pmol/l/h) and there may therefore have been a greater number of
alternative substrates present other than those added during the enrichment procedure.
Increased biomass addition would also impact the amount of DOC (dissolved organic
carbon) available in these enrichments; as bacterial and algal cells lyse and release
labile substrates. Enrichments that actively degraded CH3X also came from stations 7
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to 11. This could also be due to the fact that a greater inoculum density would
increase the likelihood of a sufficient initial CH3X-utilising population being present
to be stably maintained in the enrichment. It is also likely that the greater abundance
of phytoplankton and higher production of these stations (see Appendix C,
AMBITION cruise data) as compared to the more oligotrophic southern stations
resulted in higher local concentrations of CH3Br in the water column, particularly at
the chlorophyll maximum and above.
Enrichments were also screened for the presence of cmuA by the PCR. A number of
enrichments proved positive; see Chapter 6 for details of this investigation.
The failure of the Microbacterium isolate MJC10 to oxidise CH3Br was
disappointing, but unsurprising. Only a single Gram positive bacterium capable of
utilising CH3X as sole carbon and energy source has been previously isolated, strain
SAC4, from forest soil, which shared 98 % 16S rRNA identity with Nocardiodes
simplex. All other isolates have been members of the α proteobacteria. It might be
possible to test the hypothesis that the cmuA sequence amplified belonged to a low
abundance contaminating organism by carrying out a population fingerprinting
technique such as 16S rRNA TRFLP or DGGE, although the sensitivity of the
technique might be a problem. FISH with Microbacterium and Eubacterial probes
might also identify a contaminating organism at low abundance. The sequence itself
was identified of being a common clade of cmuA sequences that do not yet have a
cultured representative.
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Generally speaking, enrichments with CH3Br from a wide range of different marine
environments have demonstrated biological degradation of the compound. The ability
to utilise CH3Br was strongly dependent on its aqueous phase concentration, with
concentrations higher than ~300 µM being inhibitory in enrichment cultures (Hoeft et
al., 2000; Schaefer et al., 2005). As calculated aqueous phase concentration is
dependent on the Henry’s law constant used, the temperature and the volume of
headspace/volume of media ratio (See Appendix B for Henry’s law calculations), it is
important to ensure that this is carefully controlled. In situ concentrations of CH3Br
are orders of magnitude less than 300 µM (see Chapter 3) and inhibition is unlikely to
occur. It would have been interesting to investigate further the amount of biomass
produced from CH3Br in enrichments and compare these to the predicted biomass
yields using the method of (Heijnen & Van Dijken, 1992).
Since this work a number of novel marine CH3X-utilising strains have been isolated
(Schaefer et al., 2005) from marine enrichments originating from L4 seawater and
Scottish coastal water, belonging to three clades, although none of them are members
of the clades found in these enrichments (see Chapter 6 for more detail).
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Chapter 5
Methanol Dehydrogenase as a
Functional Genetic Marker
101
5 Chapter 5: Methanol Dehydrogenase as a Functional Genetic Marker
5.1 Introduction
Methanol dehydrogenase (MDH) is a pyrroloquinoline quinone-linked (PQQ-linked)
enzyme which catalyses the oxidation of methanol to formaldehyde in all Gram-
negative methylotrophs and methanotrophs that have been studied (McDonald &
Murrell, 1997). It is distinct from the alcohol dehydrogenases of Gram-positive
methylotrophs (such as Bacillus methanolicus, de Vries et al., 1992) and
methylotrophic yeasts (such as Pichia pastoris, Cregg et al., 1989). The oxidation of
methanol is a central metabolic step in Gram-negative methylotrophs, as
formaldehyde is the intermediate of both assimilative and dissimilative metabolism
(Anthony, 1982). In methanotrophs MDH is the second enzyme in the methane
oxidation pathway and oxidises the methanol produced by either the soluble or
particulate methane mono-oxygenases (sMMO and pMMO respectively).
The X-ray structure has been determined for the MDH from Methylobacterium
extorquens (Ghosh et al., 1995) and Methylophilus methylotrophus W3A1 (Xia et al.,
1996). It has an α2β2 tetrameric structure, with each α subunit containing one PQQ
molecule and one Ca2+ ion. The α subunit is approximately 66 kDa in size and the β
8.5 kDa. The α subunit has a propeller fold making up a superbarrel of eight radially
arranged β-sheets (see Fig 5.1 a). The β subunit forms a long α-helix (see Fig 5.1 b).
The genetics of methanol oxidation have been extensively studied in
Methylobacterium extorquens strain AM1 with 25 genes demonstrated to be involved
in the process, (Zhang & Lidstrom, 2003) over 5 gene clusters. Two of these encode
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a
b
c
the MDH structural genes mxaF and mxaI, which respectively encode the α and β
proteins. A third gene, mxaG encodes a specific cytochrome cL, which is the primary
electron acceptor for MDH. The mxa genes are present in Methylobacterium
extorquens AM1 as a single operon mxaW, F, J, G, I, R, S, A, C, K, L, D, E, H and B
whose expression is controlled by the M. extorquens σ70 orthologue (Zhang &
Lidstrom, 2003). The arrangement of the genes in the cluster is also conserved in
other methanotrophs and methylotrophs.
Fig 5.1. a. α subunit of MDH with coordinated PQQ and Ca2+. b. β subunit of MDH. c. α2β2 structure of MDH. Structures downloaded from http://www.ncbi.nlm.nih.gov and redrawn with Cn3D 4.1 available free from http://ncbi.nih.gov/Structure/CN3D/cn3d.shtml.
5.1.1 MDH as a functional genetic marker
There are a large number of genes involved in methanol oxidation and hence a large
number of candidates with the potential to be used as a functional genetic marker for
103
methanol oxidation. The best studied of these, and that which has been applied most
widely, is mxaF as it has regions of significant conservation, allowing ease of primer
design separated by less conserved sequence which provide the information to
construct phylogenetic analyses of the gene. The phylogeny of mxaF follows that of
16S rRNA phylogeny within those organisms that possess one and has been used as a
functional alternative to 16S based phylogenetic markers (see Fig 5.4).
McDonald & Murrell, 1997 developed a set of PCR primers mxaF1003/mxaR1561
(primer numbering throughout follows the numbering of mxaF from
Methylobacterium organophilum strain XX) based on the three complete mxaF
sequences available at the time, Methylobacterium extorquens strain AM1,
Methylobacterium organophilum strain XX and Paracoccus denitrificans. The
primers cover several important regions of the mxaF gene encoding key functions in
the protein, including Asn 287, Asp 327, Arg 357 and Asn 420, which are part of the
active site and the tryptophan docking motifs W4 and W5 which are involved in
planar stabilisation of the structure (Ghosh et al., 1995). The primers have been
applied widely and used to assess the diversity of methylotrophs and methanotrophs
in a wide range of environments including rice plant roots (Horz et al., 2001),
methane seeps (Inagaki et al., 2004), deep-sea sediment (Wang et al., 2004),
agricultural soil (Fjellbirkeland et al., 2001), and a chemoautotrophic cave ecosystem
(Hutchens et al., 2004).
One noticeable exception to the extensive use that has been made of the primers in
other environments is the paucity of information from pelagic marine systems. There
are a number of marine methylotrophic and methanotrophic isolates (Jeong et al.,
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2002; Waechter-Brulla et al., 1993; Chang et al., 2002; Lidstrom, 1988; Sieburth et
al., 1987), but relatively little information in comparison with environments such as
freshwater and soil, on mxaF diversity. A study looking for type II marine
methanotrophs, Rockne & Strand, 2003 focussed on using 16S rRNA gene PCR
primers rather than the available mxaF primers, which would have been suitable for
the purpose.
Since the original primer design (McDonald & Murrell, 1997) a number of further
complete mxaF sequences have been submitted to the database. This includes that of
Methylococcus capsulatus (Bath), which has been the subject of a genome-sequencing
project run by the University of Bergen and The Institute for Genomic Research.
Owing to the limited number of sequences used in the original primer design and the
fact that none of them belonged to the γ proteobacteria it was decided that they would
benefit from re-design in order to ensure that as full a diversity of sequences as
possible was detected in target environments.
Methyl halide degrading organisms Methylobacterium chloromethanicum strain CM4
and Hyphomicrobium chloromethanicum strain CM2 have both been demonstrated to
be capable of growth on methanol as sole carbon and energy source and also to
possess a MDH. It was proposed that analysis of mxaF sequences in methyl halide
degraders could provide a functional gene alternative to 16S rRNA phylogenetic
analysis, allowing finer resolution of relatedness as functional genes tend to mutate at
a higher rate than the 16S rRNA gene.
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5.2 Sequence availability
It was decided, as was the case in the original study (McDonald & Murrell, 1997) to
use only alignments of complete mxaF sequences available at the time rather than
partial sequences in the database, most of which had been produced using the
mxaF1003/mxaR1561 pair. The advantages of using only these sequences included
the fact that any part of the sequence can be used to design primers, rather than just
that portion already covered by the current primer pair. Disregarding sequences
amplified by the original pair avoided the tendency to develop primers that merely
amplified a subset of known sequences. The complete mxaF sequences used for
primer design are listed in Table 5.1. The sequence of the Methylococcus capsulatus
(Bath) genome had recently become available and the accession number in table 5.1
refers to it. Live Bruseth of the University of Bergen, Norway made available the
mxaF sequence of M. capsulatus (Bath) used for the alignments prior to release of the
genome sequence. This was obtained after new primers had been designed and so
they were re-designed slightly to take the new sequence and alignments into account.
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Genbank Accession
Organism Taxonomic affiliation
Reference
AF220674 Methylobacterium nodulans ORS2060
α-Proteobacteria (Sy et al., 2001)
M17339 Paracoccus denitrificans
α-Proteobacteria (Harms et al., 1987)
M31108 Methylobacterium extorquens AM1
α-Proteobacteria (Anderson et al., 1990)
M22629 Methylobacterium organophilum XX
α-Proteobacteria (Machlin & Hanson, 1988)
U41040 Methylophilus methylotrophus W3A1
β-Proteobacteria (Xia et al., 1996)
AF184915 Methylovorus sp. SS1 β-Proteobacteria (Bulygina et al., 1993)
AB004097 Hyphomicrobium methylovorum GM2
α-Proteobacteria (Tanaka et al., 1997)
AE017282* Methylococcus capsulatus BATH
γ-Proteobacteria (Ward et al., 2004)
Table 5.1. mxaF sequences used for primer development.
5.3 Alignments and primer design
The alignments were constructed using the MegAlign package from the DNAStar
suite of programs. From this candidate primers were designed by hand, looking
particularly at regions outside the current primer region so as to maximise sequence
length for phylogenetic analysis, whilst also covering the aforementioned conserved
regions. This also allowed comparison of sequences produced using the new pair
with the substantial number in the Genbank database produced with the original
primer set. Potential forward and reverse primers that were designed are listed in
Table 5.2 along with the sequences of the original primer pair and in the alignments of
Fig 5.2. Phylogenetic analysis of the sequences used in primer design can be seen in
Fig. 5.4.
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Primer Sequence 5’-3’ Reference mxaF1003 GCGGCACCAACTGGGGCTGGT (McDonald & Murrell,
1997) mxaF1013 YTGGGGYTGGTAYGCCTAYGA This thesis mxaF1080 TGGAACGARACCATGCGTCC This thesis mxaF1101 GGCGACAACAAGTGGACSATG This thesis mxaR1561 GGGCAGCATGAAGGGCTCCC (McDonald & Murrell,
1997) mxaR1476 CCCTGGTTGTGRWARCCCAT This thesis mxaR1555 CATGAABGGCTCCCARTCCAT This thesis mxaR1590 GCRCCAACRAAGAACTGGCC This thesis mxaR1590V GCRCCVACRAAGAACTGVCC This thesis Table 5.2. mxaF primers used in this study. A further forward primer mxaF909 was also designed, but this was rejected after receipt of the Methylococcus capsulatus (Bath) mxaF sequence as it had significant mismatches. Primer numbering is based, as in McDonald & Murrell, 1997, on that of Methylobacterium organophilum XX. Primer mxaR1590 was tested in two versions, one with maximum redundancy (mxaR1590V) and one with minimum redundancy.
108
109
Fig. 5.2. Alignments of designed primers against the mxaF sequences used for their design. Sequences are referred to by their strain names except Paracoccus denitrificans, which is designated as Pden. Reverse primers are presented as their reverse complement and 3’ to 5’. Mismatches in any strain are in grey. With mxaR1590/V grey indicates a mismatch in mxaR1590 only and light grey indicates a mismatch in both primers.
110
5.4 Primer testing
Primers were initially tested in all possible combinations of pairs with a representative
of each of the α-, β- and γ-proteobacterial methylotrophs. Further testing of the
primers capable of amplifying these was carried out using a wide range of different
methylotrophs, including those without a methanol dehydrogenase. The PCR was
carried out with hot start at 94oC for 5 min, followed by 30 cycles of 1 min each
denaturing at 94oC, annealing at 55oC and amplification at 72oC, a final extension step
of 10 min at 72oC was included. The PCR mixes were formulated as per the standard
mix in the methods section.
5.4.1 Initial testing
Methylobacterium extorquens AM1 (α-proteobacteria), obtained from the University
of Warwick culture collection, Methylophilus methylotrophus W3A1 (β-
proteobacteria) obtained from Prof. Nigel Scrutton, University of Leicester and
Methylococcus capsulatus (Bath) (γ-proteobacteria) were used to test the primers on
mxaF containing members of each of the α, β and γ proteobacteria. Results are as
Table 5.3.
mxaR1561 mxaR1476 mxaR1555 mxaR1590 mxaR1590V mxaF1003 558 473 552 587 587 mxaF1013 548 463 αγ 542 577 α 577 mxaF1080 481 396 α 475 α 510 510 mxaF1101 460 375 αγ 454 489 α 489 Table 5.3. Expected product sizes for each of the combinations of primer and results of the first trials. For each primer pair product size in bp is shown followed by any organism not amplified by that pair with α indicating, Methylobacterium extorquens AM1, β indicating Methylophilus methylovorus W3A1 and γ indicating Methylococcus capsulatus (Bath). Primer sequences as table 5.2.
Non-specific amplification could be seen with certain primer pairs and templates.
mxaF1101 demonstrated non-specific products with all reverse primers and both α
and β templates, as did mxaF1080. The original primer pair in combination produced
111
some non-specific amplification with the β template. Conversely mxaF1013 did not
show any non-specific amplification when paired with any of the reverse primers.
Taking all these factors into consideration it was decided to investigate further primer
pairs mxaF1003/mxaR1476, mxaF1003/mxaR1555, mxaF1013/mxaR1561,
mxaF1013/mxaR1555, and mxaF1013/mxaR1590V.
5.4.2 Further testing and primer selection
The five candidate primer combinations were next used to amplify mxaF products
from environmental DNA samples. Two contrasting sets of samples were used, four
samples taken from soil and water in Movile cave, an enclosed cave ecosystem with a
1-2 % methane atmosphere in Romania (Hutchens et al., 2004), and six samples of
total marine DNA from Eilat, Israel. Selection of primer pairs was based on
amplification from the largest number of samples and again based on a lack of non-
specific binding. mxaF1003/mxaR1555 and mxaF1013/mxaR1555 worked well based
on these criteria, although products were only obtained from two of the four Movile
cave samples and only faint products obtained from the Eilat marine samples (see Fig
5.3).
112
Fig 5.3. mxaF PCR of a range of environmental samples. The two unmarked lanes contain Invitrogen 1 kb ladder. Lanes 1-14 are respectively: Negative control; M. capsulatus BATH; M. methylotrophus W3A1; M. extorquens AM1; Eilat marine samples lanes 5-10; Movile cave samples 11-14.
Amplification by these primer pairs was checked with a wide range of methylotrophs
and methanotrophs and DNA samples from non-mxaF containing methylotrophs were
used in order to check specificity. Table 5.4 lists the genomic DNA used in these
tests. All were amplified with 16S rRNA primers f27 and r1492 (Lane, 1991) and the
products sequenced to check the identity of the DNA sample. The mxaF products
were also sequenced for the same purpose.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
113
Organism Phylogenetic affiliation Known to possess mxaF Methylosinus trichosporium OB3b
α-Proteobacteria Yes
Methylosinus sporium 5 α-Proteobacteria Yes Methylocystis parvus OBBP
α-Proteobacteria Yes
Hyphomicrobium sp.P2* α-Proteobacteria Yes Methylobacterium sp.P3* α-Proteobacteria Yes Ancylobacter sp. SC5.10* α-Proteobacteria Yes Methylobacterium sp. PM1*
α-Proteobacteria Yes
Afipia felis 25E-I† α-Proteobacteria Yes Methylophilus sp. ECd4* β-proteobacteria Yes Ralstonia sp. EHg5* β-proteobacteria No Methylococcus capsulatus BATH
γ-Proteobacteria Yes
Methylomonas methanica S1
γ-Proteobacteria Yes
Methylomonas rubra γ-Proteobacteria Yes Methylomicrobium album BG8
γ-Proteobacteria Yes
Methylomonas agile A20 γ-Proteobacteria Yes Pseudomonassp. PM2* γ-Proteobacteria No Flavobacterium sp. RD4.3*
Bacteroidetes No
Rhodococcus sp. RD6.2* High G+C Gram positive No Mycobacterium ratisbonense EM3*
High G+C Gram positive No
Arthrobacter sp. SK1.18* High G+C Gram positive No Table 5.4. Genomic DNA samples used for testing efficacy of primer pairs. All DNA was obtained from the Warwick Culture Collection (prepared by Hanif Ali), except for *, which were from Dr. Paulo De Marco, from the University of Porto, Portugal, and †, which was from Dr. Azra Moosvi from King’s College, London.
Primer pair mxaF1013/mxaR1555 failed to amplify products from Methylomonas
agile A20, but mxaF1003/mxaR1555 gave the expected products with all mxaF-
containing organisms and no products with those that do not contain mxaF.
The only exception to this was with Pseudomonas PM2, which gave an mxaF product
despite the fact that this organism is believed to contain an ExaA (Pacheco et al.,
114
2003), a PQQ-linked ethanol dehydrogenase which can be found in Pseudomonas
aeruginosa ATCC 17933 (Groen et al., 1984) and Pseudomonas putida KT2440
(Pacheco et al., 2003). On analysis of the sequenced 16S rRNA gene PCR product
from this DNA sample it was found to be that of an Ancylobacter, indicating possible
cross contamination with DNA from another sample. The original strain was
obtained and the PCR repeated, with the same result, indicating potential
contamination of the strain at source. Only a faint product was obtained with
mxaF1003/mxaR1555 and Afipia felis 25E-I, upon re-amplification with the same
primer set it was successfully sequenced and confirmed by comparison with the
Genbank sequence AY848826.
When comparing mxaF1003/mxaR1555 with mxaF1003/mxaR1561 it was found that
they amplified the genomic DNA samples identically, with the exception of that of
Afipia felis 25E-1. Upon comparing the mxaF sequence AY848826 with the sequence
of the primers it was noted that there are no mismatches with primer mxaR1555.
There are also no mismatches with all but the first base (5’ to 3’) of mxaR1561, which
is not covered by the sequence. It is impossible to ascertain whether there are any
mismatches with mxaF1003 as the sequence does not cover this area of the gene. In
their publication, Moosvi et al., 2005 report that they were able to amplify mxaF from
two of four methylotrophic Afipia isolates, including that of A. felis strain 25E-1 using
primer pair mxaF1003/mxaR1561 in contrast to the findings presented here. It is
possible that the non-amplification in this study was due to the template quality as two
amplifications were required for primer pair mxaF1003/mxaR1555 before there was
sufficient product for sequencing.
115
Fig 5.4. Maximum likelihood tree of mxaF and xoxF DNA sequences using the AxML program of ARB. Positions included in the analysis corresponded to nucleotides 922-1299 of Methylobacterium organophilum XX and the non-specific PQQ-linked alcohol dehydrogenase of Pseudomonas aeruginosa was used as an outgroup. Bootstrap values from parsimony analysis are indicated on the tree by closed circles (>95 %) and open circles (75-95 %). Species in bold were used for primer design. A = α-Proteobacterial methanotrophs, B = α-Proteobacterial methylotrophs, C = γ-Proteobacteria, D = β-Proteobacteria, E =xoxF sequences.
A
B
C
D
E
116
5.4.3 XoxF
XoxF is a putative PQQ-containing dehydrogenase found in Paracoccus denitrificans,
which has a certain level of similarity with MxaF at both the protein and DNA levels.
Similar putative dehydrogenases have been found in Methylobacterium extorquens
AM1 where it is known as MxaF’ and non methanol-utilising bacteria such as the
Rhizobia (Sy et al., 2001). With the position and number of mismatches present in
the primer binding regions non-specific amplification of xoxF was an unlikely
possibility. In order to confirm that the primers were specific for mxaF genomic
DNA from Methylobacterium extorquens AM1, which possesses both
dehydrogenases, was PCR amplified using mxaF1003 and mxaR1555. The restriction
enzyme MboII with target sequence 5’-GAAGAN8-3’ was found by computer
analysis using ARB (Ludwig et al., 2004) to be able to cut xoxF sequences and not
mxaF sequences. Digestion of the PCR product from M.extorquens resulted in the
expected banding pattern for mxaF products, with no contamination from xoxF.
5.5 Beijerinckia mobilis
Recently (Dedysh et al., 2005) published primers for the amplification of mxaF from
Beijerinckia mobilis, a heterotrophic nitrogen-fixing bacterium, previously unknown
to be capable of methylotrophy that they demonstrated to be capable of
methylotrophic growth on methanol. They were unable to obtain mxaF products
using the mxaF1003/mxaR1561 primer combination from B. mobilis, Albibacter
methylovorans DSM 13819, or Methylophaga marina ATCC 35842 and so designed
new primers, mxaF-f769, mxaF-r1392, mxaF-r1585 and mxaF-r1690. Amplification
of products from A. methylovorans was only possible with mxaF-f769 and mxaR1561,
but they report that most consistent amplification was obtained with the mxaF-f769
117
and mxaF-r1690 primer combination. The primers were designed to alignments of
the complete mxaF sequences of α Proteobacteria only and demonstrate a number of
critical mismatches with β and γ Proteobacteria, rendering these primers unsuitable
for use as functional genetic markers of methylotrophy in environmental samples.
The presence of new mxaF sequences that cover the mxaF1003 primer region in the
Genbank database allowed checking of this sequence to see whether it could be at all
improved upon. Few mismatches are introduced when alignments include the
Genbank sequences released by Dedysh et al (2005; AJ878068, AJ878070,
AJ563936, AJ878069, AJ878071, AJ878072, and AJ878073), and none of them in
positions likely to cause non-amplification of sequences. In order to be as inclusive
as possible mxaF1003 could be altered with two new redundancies to result in
mxaF1003Y, reading 5’ GCGGCACYAAYTGGGGCTGGT 3’ (see also Fig. 5.4.).
5.6 Discussion and future work
The primer pair mxaF1003/mxaR1555 show promise for use in studies of diversity of
both methylotrophic and methanotrophic organisms. The main advantage over the
current primer set is that they have been designed from a phylogentically more
diverse set of mxaF sequences and should therefore be capable of picking up a wider
range of sequences from environmental samples.
One of the aims of the work was to use the primers to amplify mxaF from marine
samples and carry out analyses on these such as clone libraries. Ideally it would have
been extremely useful to amplify mxaF products from a single sample using the
original primer pair and the newly developed pair, creating equal clone-libraries and
118
comparing the diversity of sequences in them using statistical methods, such as those
provided by the LIBSHUFF program (Singleton et al., 2001) and
http://www.arches.uga.edu/~whitman/libshuff.html) in order to prove the new set are
an improvement on the old. I was unable to complete this work within the duration of
the project, as the work with cmuA and MeBr measurements took priority. Currently
within the lab Dr. Josh Neufeld is using the new primers with marine samples and
planning to use them along with the Stable Isotope Probing technique (Radajewski et
al., 2000) in order to characterise marine methanol degrading bacteria.
119
Chapter 6
Diversity of cmuA in Marine
Environments: Clone library and TRFLP
analysis
120
Chapter 6: Diversity of cmuA in Marine Environments and TRFLP analysis 6.1 Introduction CmuA is a bifunctional protein with methyltransferase and corrinoid-binding domains.
It is the first enzyme in the CH3Cl degradation pathway elucidated in Methylobacterium
chloromethanicum CM4, an organism that is capable of growth on CH3Cl and CH3Br as
sole carbon and energy source (Vannelli et al., 1998; Vannelli et al., 1999; see Fig 6.1).
Fig 6.1. Pathway of CH3Cl degradation in Methylobacterium chloromethanicum CM4 as Vannelli et al., 1999. Numbering refers to enzyme for that particular step in the pathway: 1. CmuA, methyltransferase/corrinoid protein; 2. CmuB, methyltransferase; 3. MetF, 5,10-methylene-tetrahydrofolate reductase; 4. and 5. FolD, 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (4./5.); 6. PurU, 10-formyl-tetrahydrofolate hydrolase; 7. FDH, Formate dehydrogenase.
Carbon assimilation via serine cycle
CH3Cl
H4folate
CH H4folate
CHO H4folate
CH2 H4folate
CH3 H4folate
HCOOH
CO2
H2O
H2O
H4folate
2 H+
2 H+
2 H+
HCl
CoI
CH3 CoIII
1.
2.
3.
4.
7.
6.
5.
121
This pathway is believed to be responsible for the degradation of CH3X in other
organisms capable of growth on these compounds as sole carbon and energy sources,
such as Aminobacter ciceronei IMB1 and Aminobacter lissarensis CC495 (McDonald
et al., In Press; Woodall et al., 2001). In Hyphomicrobium chloromethanicum strain
CM4 it has been shown that cmuA- mutants are no longer capable of growth on either
CH3Cl or CH3Br as sole carbon and energy sources (Borodina et al., 2004). Schaefer et
al., 2005 isolated 13 marine CH3X-utilizing bacteria belonging to three distinct clades,
two of these clades, represented by strains 179 and 198, probably make use of the
CmuA methyltransferase pathway. The third clade and another marine CH3X-utilizing
organism, Leisingera methylohalidivorans strain MB2 (Schaefer et al., 2002), are likely
to make use of a hitherto uncharacterised pathway of CH3X degradation, seemingly not
involving CmuA since there is no evidence for presence of cmuA/CmuA in these methyl
halide degraders.
CmuA is the most conserved of the enzymes in the pathway as demonstrated by derived
protein and DNA alignments between cmuA sequences from the organisms
demonstrated to contain the pathway (McDonald et al., 2002). The arrangement of the
genes of the cmu (chloromethane-utilisation) cluster is also highly conserved between
most of these organisms, with the exception being M. chloromethanicum CM4 in which
the cmu cluster is split into two sub-clusters (see Fig 6.2).
All these factors make cmuA an ideal candidate for use as a functional genetic marker to
investigate the presence and diversity of methyl halide utilisers in the environment that
use this pathway. It has been used as such in two separate DNA-stable isotope probing
experiments with soil samples enriched with either 13CH3Br or 13CH3Cl (Miller et al.,
2004) or 13CH3Cl (Borodina et al., 2005).
122
Fig 6.2. Comparison of cmu gene clusters sequenced to date. Genes involved in the metabolism of CH3X are in blue, with cmuA in red. Genes not directly involved are in green. Organisms are referred to by their strain names; Methylobacterium chloromethanicum CM4, Aminobacter lissarensis CC495, Aminobacter ciceronei IMB1 and Hyphomicrobium chloromethanicum CM2. Strains 198 and 179 are affiliated to the Roseobacter clade of the α proteobacteria, within the Rhodobacteracaea family.
6.1.1 PCR primers for cmuA
McAnulla et al., 2001 designed PCR primers for the amplification of cmuA based on
regions of conservation in alignments of cmuA from M. chloromethanicum CM4, and H.
chloromethanicum CM2 using the CODEHOP (consensus-degenerate hybrid
oligonucleotide primers) program (Rose et al., 1998) and
http://bioinformatics.weizmann.ac.il/blocks/codehop.html). The primers were designed
with the forward primer located in the 5’ methyltransferase domain and the reverse
primer located in the 3’ corrinoid-binding domain. As this is a unique structural
arrangement, the rationale was that this would increase primer specificity and not PCR
amplify methyltransferase genes or genes containing sequences coding for corrinoid-
str. IMB-1
2 kb
II
I
str. CC495
str. CM4
str. 198
cmuB cmuC cmuA fmdB paaE hutI
cobQ cobD metF cmuB cmuC cobC
cobU folC folD purU cmuA
cmuC cmuA fmdB paaE hutI metF
cmuA fmdB paaE hutI cmuC nrdF nrdA
str. CM2
cmuB cmuC cmuA fmdB paaE hutI metF //
str. 179 cmuA fmdB paaE hutI metF
123
binding regions of other polypeptides. The primers 929f and 1669r were used to
amplify successfully a 741 bp PCR product from the two isolates used to design the
primers, newly isolated Hyphomicrobium strains and from a soil enrichment culture.
Warner, 2003 designed new PCR primers using alignments of cmuA sequences from M.
chloromethanicum CM4 and H. chloromethanicum CM2 and the recently cloned cmuA
sequences of Aminobacter ciceronei IMB1 and Aminobacter lissarensis CC495. Five
forward primers and four reverse primers were designed and primer pair
cmuAF802/cmuAR1609 was selected as the best candidate. Again this spanned the two
parts of the cmuA gene encoding two functional domains of CmuA. It was this primer
pair that was used in the two previous DNA-SIP studies (Borodina et al., 2005; Miller et
al., 2004) and was selected for use in this study.
6.2 Enrichment diversity
In order to study the diversity of cmuA in the most active marine enrichments (see
Chapter 4) 2 mL of each enrichment was centrifuged for 5 min at 13 000 rpm, the
supernatant removed and the pellet resuspended in 10 µl of sterile deionised water. This
was then boiled for 10 min in a water bath and 1 µl was used as template in PCR
reactions. PCR products were visualised on 1 % agarose (w/v) gels after EtBr staining.
Bands corresponding to the expected size of product (807 bp) were excised and the
DNA was purified using the Qiaquick Gel extraction kit (Qiagen). Clone libraries of 50
to 100 clones were constructed using the Invitrogen TOPO cloning kit. Clone libraries
were dereplicated using RFLP analysis with double digests of both EcoRI/DdeI and
EcoRI/RsaI and grouping of the clones into OTUs (operational taxonomic units) was
based on the RFLP patterns produced (Fig 6.3). OTUs were determined throughout on
a per library basis.
124
Fig. 6.3. Example EcoRI/DdeI RFLP digest of cmuA clones from enrichment L4.1.The starred lane contains Invitrogen 1 kb sizing ladder. Representative clones from each OTU were selected for bi-directional DNA sequencing
using the M13 primers for the TOPO vector. In the case of the longer
cmuAF229/cmuAR1609 inserts, additional forward and reverse sequencing reactions
were carried out using cmuAF802 and its reverse complement in order to get full
sequences.
The PCR was performed using both cmuAF802/cmuAR1609 and
cmuAF229/cmuAR1609, partly in order to compare the efficacy of the two forward
primers and partly in order to try and obtain longer inserts for sequence analysis.
Primer pair cmuAF229/cmuAR1609 covers more of the methyltransferase region of
cmuA and provides much more information about the sequence diversity in this region.
6.2.1 English Channel enrichments
Three 300 mL enrichments of seawater obtained from L4, a sampling station off the
coast of Plymouth, set up at different times of the year and enriched with 0.2 % CH3Br
were analysed by cmuA PCR as above. cmuA PCR products were obtained from L4.1,
L4.2 and L4.3 with cmuAF802/cmuAR1609, but not with cmuAF229/cmuAR1609 and a
clone library was produced from enrichment L4.1. This enrichment had been
established for the longest length of time (~8 months) and had had 5 pulses of CH3Br ,
equivalent to approximately 312 µmoles of CH3Br. It also gave the brightest product of
*
125
the three and was therefore considered the best for clone library production (see Fig
6.4).
Fig 6.4. cmuAF802/cmuAR1609 PCR products from enrichments. Lane 1 is the negative control and lane 2 the positive (H. chloromethanicum CM2). The remaining lanes 2-9 are, respectively: L4.1; L4.2; L4.3; 165; 249; 249.2; PE2 refer to Chapter 4 for details of the sources of these enrichments. Clones grouped into 2 OTUs with 73 % in OTU 1 and 27 % in OTU 2. The groupings
for this particular library are based solely on EcoRI/DdeI digests as RsaI failed to cut
any of the clones. Upon construction of phylogenetic trees, all the clones from this
cmuA library formed part of a single clade (B2 in Fig 6.5) most closely related to cmuA
sequences from Aminobacter ciceronei IMB-1 and Aminobacter strain TW23, both
isolated from woodland soils, and an uncultured soil cmuA clone, ‘chloromethane-
utilising bacterium’ 2 (AF307140, McAnulla, 2000).
6.2.2 Arabian Sea enrichments
Enrichments from the Arabian Sea AMBITION cruise were set up from concentrated
seawater samples added to tenth strength marine ANMS (ammonium nitrate mineral
salts). See Chapter 4 for details.
DNA from all the enrichments amplified with cmuAF802/cmuAR1609 gave cmuA PCR
products of varying band intensities and two of these were selected for clone library
analysis, library 27 from the pooled enrichment PE2, and library 25 from the Station 10
1 2 3 4 5 6 7 8 9
126
enrichment 249. Only DNA template from enrichment PE2 gave a cmuA PCR product
with cmuAF229/cmuAR1609 and this was also selected for clone library analysis as a
comparison with the other primer set.
The enrichment 249 cmuA clone library gave 6 OTUs, which are summarised in Table
6.1.
OTU Number of clones
% of total clones
Sequenced representatives (Genbank accession no.)
1 20 17.5 E25.21 (DQ090686), E25.2 (DQ090687) 2 87 76.3 E25.4 (DQ090685), E25.11 (DQ090690) 3 4 3.5 E25.5 (DQ090684) 4 1 0.9 E25.16 (DQ090688) 5 1 0.9 E25.45 6 1 0.9 E25.139 (DQ090689) Table 6.1. OTUs from library 25, the cmuA clone library from enrichment 249. According to phylogenetic analysis, the OTUs above split into three clades, OTUs 1, 2
and 6 form a novel clade of cmuA sequences, currently without a cmuA sequence from
an isolated representative. OTU 3 groups with Aminobacter ciceronei strain IMB-1 and
Aminobacter strain TW23. E25.16, the sole member of OTU 4, is consistently placed,
independently of tree calculation method, as related to, but separate from the same
Aminobacter cmuA cluster. It shares 94.6 % identity with cmuA from A. ciceronei strain
IMB1.
The cmuA Library 27 was constructed with DNA from pooled methylotrophic
enrichments that had been grown on a variety of different carbon sources and then
enriched with CH3Br. The PCR with primers cmuAF802/cmuAR1609 gave the most
intense PCR product of any of the enrichments, which is likely to reflect the increased
biomass in the enrichment due to the pre-enrichment step. Seven OTUs were apparent
upon RFLP analysis and are summarised in Table 6.2.
127
OTU Number of clones
% of total clones
Sequenced representatives (Genbank accession no.)
1 94 94.0 E27.1, (DQ090683), E27.2, (DQ090682), E27.3, (DQ090679), E27.4 (DQ090676)
2 1 1.0 E27.22, (DQ090681) 3 1 1.0 E27.24, (DQ090680) 4 1 1.0 E27.30, (DQ090678) 5 1 1.0 E27.32, (DQ090677) 6 1 1.0 E27.45 7 1 1.0 E27.48, (DQ090675) Table 6.2, OTUs from library 27, the cmuA clone library from enrichment PE2.
OTUs 1, 2, 3, 5, 6, and 7 form a single marine clade (A3) in cmuA phylogenetic trees.
The closest cmuA sequences from cultured CH3Br-utilsing bacteria being
hyphomicrobia. These form a separate clade with DNA sequence identity of 81.1 %
and 77.4 % respectively to cmuA sequences from Hyphomicrobium strain LAT3 and H.
chloromethanicum CM2 (E27.3 used for distance calculation). E27.22 (OTU 2),
E27.24 (OTU 3) and E27.48 (OTU 7) cmuA sequences consistently branch more deeply
than the other members of this clade, which could account for them being placed into
separate OTUs by RFLP analysis. E27.30 (OTU 4) cmuA sequence grouped with the
novel clade (B2) of marine cmuA sequences formed by OTUs 1 and 2 of the Station 10
enrichment cmuA library.
Library 9 contained the longer PCR product cmuA sequences from primer pair
cmuAF229/cmuAR1609 and the same template as for library 27. The library was
dereplicated using RFLP analysis with the same restriction enzymes as for the other
libraries. With the increased length of the cmuA sequences, this provided a finer level
of discrimination than that for the other libraries, which should be borne in mind when
examining the OTUs produced (Table 6.3).
128
OTU Number of clones
% of total clones
Sequenced representatives (Genbank accession no.)
1 58 75.3 E9.8, (DQ090668), E9.1, (DQ090674) 2 1 1.3 E9.3, (DQ090670) 3 10 14.2 E9.22, (DQ090673), E9.28, (DQ090671) 4 2 2.6 E9.27, (DQ090672) 5 1 1.3 E9.62, (DQ090669) 6 1 1.3 E9.81, (DQ090667) 7 2 2.6 E9.91, (DQ090666) 8 2 2.6 E9.92, (DQ090665) Table 6.3. OTUs from library 9, the cmuA clone library from enrichment PE2, amplified with primers cmuAF229/cmuAR1609. Despite the number of OTUs produced, all the sequences clustered together in a single
clade (A3) upon phylogenetic analysis. This was the same clade in which the majority
of library 27 cmuA clones were found. It is interesting that no clones were identified
with similarity to the Aminobacter clade unlike library 27, despite the fact that the
template DNA was identical. This could be explained stochastically as only 1.0 % of
library 27 (a single clone) grouped in this clade, but coupling this with the fact that
primer pair cmuAF229/cmuAR1609 was unable to amplify cmuA products from DNA
from any of the other enrichments suggests that it is specific for this particular clade of
sequences. Primer pair cmuAF802/cmuAR1609 is evidently capable of picking up a
wider diversity of cmuA sequences.
6.3 cmuA clone library analysis with DNA from high volumes of Arabian
Sea samples
These samples were kindly supplied by Dr. Clare Bird and Dr. Mike Wyman of the
University of Stirling, UK. The samples were taken using stand-alone pumps (SAP;
Challenger mark 2 SAP, Challenger Oceanic, UK). These pumps are automated and
pump large volumes of water for a set period of time through large (293 mm, 0.2 µm)
filters, achieving effective water sample volumes of 36 to 200 L, during this study.
DNA samples were supplied after being extracted using the following method (Mike
129
Wyman, pers. comm.). SAP filters were rinsed in 5 ml filtered seawater and the filtrate
taken up in 1 ml RNALater (Ambion) and stored at 4 oC. 0.5 ml of this was centrifuged
and DNA isolated from the resulting pellet using a Qiagen DNA extraction kit with the
DNA eluted in 100 µl sterile deionised water. 1 µl of this was used as template for PCR
amplification of cmuA, at neat and 1:10 dilutions. The effective volume of sample
represented by 1 µl of extracted DNA can be seen in Table 6.4.
Station/cast number Depth (m) Volume sampled (L)
Effective volume sampled in PCR (mL)
01/08 30 96 80.0 02/07 45 177 147.5
03/07 20 85 70.8 04/02 20 200 166.7 05/02 15 59 49.2 06/03 10 100 83.3 07/08 220 106 88.3 08/02 20 47 39.2 09/03 20 36 30.0 Table 6.4. Effective sample volumes for 1 µl volumes of DNA extracts used as templates in amplification of cmuA PCR products from SAP samples. PCR products using primer pair cmuAF802/cmuAR1609 were obtained from the
samples from stations 1 (01/08), 2 (02/07), 4 (04/02) and 9 (09/03). The cmuA PCR
products from station 2 were smaller than the expected size of 807 bp and upon test
sequencing proved not to be cmuA. The PCR products were faint and proved difficult
to clone, therefore only small libraries of 50 cmuA clones were produced from each of
the remaining PCR products. Upon RFLP analysis with EcoRI/DdeI and EcoRI/RsaI
double digests, the station 9 cmuA library was shown to contain only a single OTU.
The same OTU made up 98 % of the station 4 cmuA library with a single representative
(S4.14) in OTU 2. The station 1 library contained two OTUs: 70 % OTU 1 and 30 %
OTU 2 (see Table 6.5) neither of which were similar to those cmuA sequences in the
other two libraries.
130
Library Number of clones
% of total clones
Sequenced representatives (Genbank accession no.)
1 (OTU 1) 35 70 S1.1, (DQ090703), S1.2, (DQ090702) 1 (OTU 2) 15 30 S1.4, (DQ090701), S1.5, (DQ090700),
S1.43, (DQ090705) 4 (OTU 1) 49 98 S4.3, (DQ090699), S4.4, (DQ090698), 4 (OTU 2) 1 2 S4.14, (DQ090704) 9 50 100 S9.1, (DQ090697) Table 6.5. OTU assignment of cmuA sequences from SAP sample clone libraries.
All the cmuA sequences retrieved from DNA from stations 4 and 9 clustered with the
highly related clade of marine cmuA sequences consisting entirely of sequences from
the two pooled enrichment libraries (A3). The station 1 derived cmuA clones grouped
with the cmuA clade formed by the station 10 enrichment cmuA library (B2).
6.4 Phylogenetic analysis
Phylogenetic trees were constructed with all available cmuA sequences. The large
number of cmuA sequences (136) combined with the length of sequence analysed (552
bp, corresponding to nucleotides 1005-1557 of Methylobacterium chloromethanicum
CM4) was towards the upper limits (150 sequences) of analysis of the ARB program
(Ludwig et al., 2004) with Maximum-likelihood analysis.
The analysis was left running for two weeks with no sign of completion, and then
aborted. Instead a parsimony tree was produced using ARB with 10 bootstraps, as
again the number of sequences involved was too large to analyse with a greater number
of bootstraps than this. Location of nodes was confirmed by Neighbour Joining
analysis using the Seqboot, Dnadist, Neighbour and Consense programs of PHYLIP
(Felsenstein, 1989; Felsenstein, 2004) with 100 bootstraps (Fig 6.5). Parsimony
bootstrap values over 75 % are marked on the tree. Major clades are labelled A1 to B4
and referred to in the text.
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Fig. 6.5. cmuA parsimony tree of 552 bp. See text for full details and discussion. Sequences obtained in this study are bold and red. Isolates are bold.
A1
A2
A3
A4
B1
B2
B3 B4
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As Maximum Likelihood analysis could not be carried out on the full data set, a
selection of cmuA sequences was analysed, representing each of the major clades (Fig.
6.6) in order to confirm the general tree topology seen in Fig. 6.5.
Fig. 6.6. cmuA Maximum-likelihood analysis performed using the same area of sequences as Fig. 6.5. Parsimony analysis bootstrap values (100 samplings) are indicated by closed circles (>95 %) and open circles (75-95 %).
Maximum Likelihood trees of cmuA sequences were also constructed for each of the
domains of the molecule, corresponding to bases 866-1124 (methyltransferase), 1210-
1571 (corrinoid-binding) and 1125-1209 (linker region) of the Methylobacterium
chloromethanicum CM4 cmuA sequence. All three trees were identical, with branching
order and relative positioning of sequences completely conserved. This gives an
indication that the sequences were not chimeric.
When carrying out BLASTp (Altschul et al., 1997) analysis of CmuA sequences it was
observed that the closest non-CmuA proteins are the Mono-, Di-, and Tri-methylamine
B4
B3
B2
B1
A4
A3
A1
A2
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methyltransferases and corresponding corrinoid-binding proteins. It was alignment with
these that allowed assignment of domains of the cmuA sequences in the above analysis.
6.5 Correlation of clades with in situ populations
In terms of enrichments, correlation of cmuA diversity with actual in situ organisms is
impossible other than indicating presence of the cmuA sequence in an organism at the
particular station. Even absence cannot be assured, as the organism indicated by a
particular sequence may have been out-competed in the enrichment. Equally, an
organism that was scarce and carrying out only a small fraction of the environmental
CH3Br oxidation may have been favoured during the enrichment process, in this case
perhaps as the CH3Br concentrations used were orders of magnitude greater than
environmental concentrations.
Examination of the sequences gathered from the three SAP sample libraries from the
AMBITION cruise are more informative, as they are more representative of the in situ
community. Station 1 derived cmuA sequences (S1_x) all clustered together within
clade B1, whilst cmuA sequences from stations 4 (S4_x) and 9 (S9_x) formed a
separate, exclusively marine clade, A3. A shift in population of cmuA containing
bacteria seems to have occurred between stations 1 and 4. Interestingly the only
enrichment to show evidence of the presence of clade B1 sequences was 249, from
station 10 (E25_x), indicating that organisms containing similar cmuA sequences are
present in both the oligotrophic and eutrophic regions of the cruise track. It is possible
that this could be a bacterial species capable of existing under the two contrasting
environmental conditions exemplified by stations 1 and 10, or that this simply
highlights a lack of correlation between cmuA sequence type and phylogeny. The
sequence type of cmuA also displays no correlation between environment, with marine
sequences from the Arabian Sea sharing high identity with sequences from soil isolates.
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6.6 Terminal restriction fragment length polymorphism analysis
6.6.1 Rationale
It seemed at this point that a wide range of different clades of cmuA sequences could be
found in the Arabian Sea DNA samples and enrichments and that it would be interesting
to investigate the location of these with respect to depth in the water column,
geographical location, and physicochemical factors. Two stations (3 and 7) had been
sampled regularly for a diel cycle, allowing investigation of diversity of cmuA
sequences at different depths in the water column in response to day/night changes.
As there were many samples to screen for the presence of cmuA, it was decided that a
rapid technique for surveying genetic diversity should be employed. There are a
number of well-established methods that would have been appropriate in this case,
including denaturing gradient gel electrophoresis (Riemann et al., 1999), terminal
restriction fragment length polymorphism (Moeseneder et al., 1999) analysis, and
length-heterogeneity PCR (Ritchie et al., 2000). The cmuA genes that had been
sequenced to date had little heterogeneity in length, which would have left L-HPCR
missing much of the potential diversity. With DGGE there is the potential to excise and
sequence the most intense bands on the gel, giving sequence information for the most
common amplicons. However, it was believed that TRFLP would prove to be the most
suitable technique as it was rapid, straight forward to develop for a new gene and
allowed easy intercomparison of samples, since standards can be run within each
sample, unlike DGGE. The large database of marine and terrestrial cmuA sequences
that had been previously collated also facilitated the development of the TRFLP
technique for assessing the diversity of environmental cmuA sequences.
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6.6.2 TRFLP
TRFLP relies on the PCR amplification of a gene using primers labelled at the 5’ end
with a fluorescent marker. The labelled PCR products produced are then digested with
restriction enzymes. The terminal restriction fragment remains labelled, whilst all other
restriction fragments are unlabelled. A fluorescent sizing ladder is added to each
sample and running the restriction-digested products on a DNA sequencer in gene scan
mode allows sizing of the terminal restriction fragments produced. The discrimination
of clades of the gene and assessment of diversity relies on careful selection of restriction
enzymes. Splitting samples and using several different restriction enzymes can increase
discrimination, as can using a second complimentary fluorescent label on the reverse
primer. Another advantage of TRFLP is that the relative fluorescence of each TRF
(terminal restriction fragment) indicates the relative abundance of the particular PCR
product in the reaction (Osborn et al., 2000). This is not wholly quantitative as
abundance of a PCR amplified gene does not reflect the abundance of that particular
sequence in the original sample, but it does indicate which clades have the highest
relative abundance.
When using a capillary sequencer it has been noted that there is a bias towards smaller
fragments as they are favoured in the electrophoresis and this can lead to an over-
estimation of the abundance of such fragments (Moeseneder et al., 1999).
6.6.3 Development
The first part of the development process involved selection of restriction enzymes that
could discriminate between the clades of cmuA sequences that had been sequenced to
date (see Fig 6.5). Restriction enzymes that recognise 4 base restriction sites are
favoured in TRFLP analysis since enzymes with longer recognition sites tend not to cut
frequently enough.
136
An ARB (Ludwig et al., 1998) database was constructed with cmuA sequences edited to
include the forward and reverse primer sequences (as the primer forms part of the TRF
it needs to be included in order to predict the correct sizes of TRFs). Sequences that had
been produced with primer pairs other than cmuAF802/cmuAR1609 were either
trimmed (as in the case of the L9 clone sequences or the complete cmuA sequences such
as that of Aminobacter ciceronei strain IMB-1) to the same length as the other
sequences, or discarded (as in the case of soil clones AF307140, AF307141 and
AF307142). This left a standardised set of 137 cmuA sequences that could be
interrogated using the probe, user1 and user2 fields of the ARB Editor window. They
allow the user to input oligonucleotide sequences which are subsequently highlighted
wherever they are found in the sequence alignment. Restriction enzymes were
discarded if they did not discriminate well between clades, cut within the primer
sequence, or produced TRFs that were outside the 35-500 bp limit of sizing of the
ladder used. Restriction enzymes screened included all 4 base cutters available from
New England Biolabs (US), and Helena Biosciences (UK), excluding isoschizomers.
The restriction enzymes BsiYI, HaeIII and HpaII were selected using these criteria.
BsiYI provided good discrimination between different clades of cmuA in both the
forward and the reverse primer directions, HaeIII discriminated at the 5’ end; HpaII
discriminated at the 3’ end. Predicted TRFs for the members of the tree in fig 6.5 can
be seen for each of the enzymes in Table 6.6. Appendix D lists the sequences
represented by each TRF.
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Enzyme Recognition site TRFs (bp) HaeIII 5’ C^CGG 34, 43, 53, 121, 145, 167, 271, 308, 479.
BsiYI 5’ CCNNNNN^NNGG 50, 126, 276 329, 346, 384, 397, 402, 461, 464,467,483, uncut.
BsiYI 3’ CCNNNNN^NNGG 114, 166, 171,282, 325, 335, 347, 391, 406, 417, 427, 764, uncut.
HpaII 3’ GG^CC 90, 110, 115, 135, 144, 156, 225, 330, 459, 481, 483, 625.
Table 6.6. TRF sizes of known cmuA sequences. Those in red are outside the sizing range possible when using the ROX 500 ladder. Uncut PCR products will be ~802 bp in length.
6.6.4 Method validation with standard cmuA clones
The TRFLP method was tested using three cmuA clones as standards. TOPO
(Invitrogen) cloned cmuA products of strain 179, and marine enrichment cmuA clones
PMLSW6 and PML1A4 (Schaefer et al., 2005) were used as templates in PCR reactions
with labelled primers. PCR reactions were carried out as described in Chapter 2,
Materials and methods. Several aliquots of Taq polymerase were pooled to provide a
source of Taq sufficient for a large number of reactions; it has been reported that
variation in the reproducibility of TRFLP and other PCR-based population
fingerprinting methods can be affected by variability in batches of Taq polymerase
(Osborn et al., 2000), even when obtained from the same supplier and this measure was
designed to remove this variability.
PCR primers were 5’-labelled with the fluorescein-derived dyes 6-FAM
(6-carboxyfluoroscein, cmuA802F) and HEX (6-carboxy-2’,4,4’,5’7,7’-
hexachlorofluoroscein, cmuA1609R, TagN, Newcastle). Primers were routinely
resuspended in 10 mM Tris HCl pH 8.0 rather than deionised water since more acidic
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pHs than this cause bleaching of the fluorophore. The remainder of sample preparation
was carried out as described by (Moeseneder et al., 1999) with one change, PCR
products were loaded onto a large welled 1 % (w/v) agarose gel, excised and gel
extracted, rather than being precipitated first and then run on an agarose gel. It was
found that this did not reduce the quality of TRFLP patterns produced and also reduced
losses of PCR product. Once samples were precipitated and dried, they were split into
three aliquots for digestion in 100 µl total volumes with each of the above restriction
enzymes. Digestion was overnight at 37 oC for HaeII and HpaII and overnight at 55 oC
for BsiYI, with 5U of enzyme used in each case. Once digested the samples were
precipitated, dried and resuspended in 4 µl HiDi formamide (Applera, UK). 2 µl of the
HiDi formamide resuspended sample was mixed with a further 10 µl HiDi formamide
and ROX 500 (5-carboxy-X-rhodamine) size standard, diluted as per manufacturer’s
instructions, and loaded onto a 3100 Genetic Analyser (Applied Biosytems). The
analysis was carried out with 36 cm capillaries, using the POP4 polymer and the
standard run time was 45 min. The machine was set with the appropriate filter for
analysis of ABI dye set D. Data were initially collected and analysed using GeneScan
software, but this was found to be erratic in ‘calling’ the correct sizes for TRFs and so
GeneMapper v3.0 was consequently used in preference.
It would have been possible to use FAM, HEX, NED and TAMRA fluorescent dyes and
to have combined the products into single sample runs; the dye-sets are complimentary
with non-overlapping emission and absorption spectra. However, it was noted that with
BsiYI samples, where the forward primer was FAM labelled and the reverse was HEX
labelled, that a certain amount of dye bleed-through was evident. Peaks were produced
in the HEX detection channel at identical places, but with less intensity, as FAM peaks.
This could potentially make resolving the exact TRF difficult, particularly in the case of
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the BsiYI 5’ TRF 346 and BsiYI 3’ TRF 347. At this time since BsiYI 5’ TRF 346 was
uncommon and part of an exclusively terrestrial clade, this problem was believed to be a
minor one and could be resolved in future by using an alternative fluorescent dye for 3’
TRFs that has emission and absorption wavelengths further removed from those of
FAM, such as NED (see Table 6.7 for dye wavelengths). Samples were therefore kept
separate rather than multiplexed in runs for the validation of the method (see Fig 6.7 for
an example TRFLP pattern and Appendix D for a full list of the TRF assignments for
each sequence).
Fig 6.7. BsiYI TRFLP pattern of clone PMLSW6 (AJ810829). The x axis is in bp and the y in relative fluorescent units. The red peaks are ROX 500 ladder, with FAM labelled TRF in blue and HEX in green. Sizing analysis was performed using the Global Southern size-calling algorithm of the Genemapper 3.0 software (Applied Biosystems, US). Fluorescent Dye Absorption maximum
(nm) Emission maximum (nm)
FAM 494 522 HEX 535 553 NED 546 575 ROX 587 607 TAMRA 560 582 Table 6.7. Absorption and Emission maxima of fluorophores for TRFLP analysis, adapted from www.appliedbiosystems.com.
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6.7 TRFLP on environmental samples
The samples taken for DNA extraction from the AMBITION cruise are listed in
Appendix A. Each was from 2 L of seawater filtered either though 0.2 µm sterivex
cartridge filters, or through 0.2 µm Supor 200 membrane filters. Initially a number of
sterivex samples were selected for DNA preparation using the method of Somerville et
al., 1989 and amplification of cmuA was attempted with primer pair
cmuAF802/cmuAR1609 (see Table 6.8 for sample list).
Station Sample (refer to Appendix A) 1 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13
2 17, 18 3 (Diel) 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 4 49, 50, 51, 52 ,53 5 66 6 82, 83 7 (Diel) 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 8 114, 115 9 122, 130, 131 10 138, 140 11 154
Table 6.8. AMBITION samples analysed by cmuA PCR. Both sterivex and memebrane filter samples are included. At least the chlorophyll maximum and surface water samples were used at all stations except 5 and 11.
Only non-specific PCR products were observed with these samples. Further DNA
samples were extracted using the hot-phenol method of (Schaefer & Muyzer, 2001)
with the membrane filtered samples. This was the preferred method of DNA sample
preparation as the Supor 200 filters are made from Polyethersulfone and are phenol
soluble; there is no chance of bacteria remaining attached to the filter and avoiding
lysis, which is a potential hazard when using the Sterivex filters. Despite repeat
attempts at PCR with a range of template concentrations cmuA products were not
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obtained from any of these samples, although 16S rRNA products using primer 27f and
1492r could be obtained.
6.7.1 Celtic Sea seawater filter samples
These samples were kindly provided by Dr. Gary Smerdon of Plymouth Marine
Laboratory and were taken during a cruise aboard RRS Discovery (D261) in the Celtic
Sea from the 1st to 14th April 2002. The aim of the cruise was to follow and sample a
spring phytoplankton bloom through its formation and subsequent breakdown.
Phytoplankton blooms have been demonstrated at sites of particularly high levels of
CH3Br (Baker et al., 1999; Wingenter et al., 2004) and as such it was hoped that
bacteria capable of degrading CH3Br would also be present. The samples taken were
500 ml of water filtered through 0.2 µm nucleopore filters (see Table 6.9). DNA was
extracted using the hot-phenol method of Schaefer & Muyzer, 2001.
Station and cast no.
Station type Date of Sampling
Lat./long. Depths sampled (m)
1.12 E1 – standard 02/04/02 50o02’N 04o22’W 2 and 40 4.2 Standard 03/04/02 49o32’N 06o00’W 2 and 40 6.3 Standard 04/04/02 48o41’N 11o12’W 5 and 40 6.12 Standard 05/04/02 48o41’N 11o12’W 2 and 42 7.36 Lagrangian 09/04/02 49o37’N 10o20’W 2 and 40 7.54 Lagrangian 10/04/02 49o37’N 10o20’W 2 and 35 7.90 Lagrangian 12/04/02 49o37’N 10o20’W 2 and 35 Table 6.9. Celtic Sea samples.
Extracted DNA was used at a range of dilutions (neat, 1:10 and 1:100) for the PCR with
primer set cmuAF802/cmuAR1609. No cmuA PCR products were observed for any of
the samples. Amplification was checked by amplification of the 16S rRNA gene using
primers 27f/1492r, which proved positive in all cases, indicating the absence in DNA
samples of compounds inhibitory to the PCR.
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6.8 Discussion and future work
Three different clades of cmuA sequences were detected during the course of this study.
One completely novel clade (A3) lacked isolated representatives, with the most closely
related sequence from isolated species being Hyphomicrobia. The Hyphomicrobia are
not known to be a marine genus, although methanol dehydrogenase sequences (mxaF)
affiliated to mxaF from Hyphomicrobium have been recovered from deep-sea
sediments, (Wang et al., 2004). The two other clades that were recovered were B1 and
B2. The cmuA sequences obtained from Arabian Sea DNA samples of enrichments and
seawater in clade B1 were distinct from the rest of the clade as supported by parsimony
and neighbour joining bootstrap values in phylogenetic analysis. They were most
closely related to cmuA sequences from a Rhodobacteracaea isolate, 179, isolated from
CH3Br enrichments of English Channel seawater, off the coast of Plymouth, indicating
a wide geographic spread of this clade of sequences. B1 also contains cmuA sequence
representatives previously amplified from soil communities (Borodina et al., 2005). All
L4 enrichment cmuA sequences clustered in clade B2, together with cmuA sequences
from isolates Aminobacter ciceronei IMB1 and Aminobacter sp. TW23, both isolated
from soils. This highlights the diversity and spread of the cmuA sequences. It would be
interesting to see whether this is reflected phylogenetically, by using a technique such
as stable-isotope probing (Radajewski et al., 2000), which has been used successfully
with both 13CH3Br and 13CH3Cl in terrestrial environments (Borodina et al., 2005;
Miller et al., 2004). An enrichment was set up with an L4 seawater sample and
13CH3Br with this intention towards the latter stages of this investigation, although it
failed to oxidise the compound.
It was interesting to discover that cmuA could not be amplified from DNA from the
smaller volume environmental samples when it could from the larger volume samples.
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This indicates something about the abundance of these organisms within the
environment. Effective sample volumes of the SAP samples were all greater than 30 ml
in each µl of PCR template. The cmuA PCR positive samples of stations 1, 4 and 9 had
effective sample volumes in each 1 µl of DNA template of 90, 166.7 and 30 ml
respectively. DNA extracts from the 2 L cruise samples had effective sample volumes
of 10 –20 ml depending upon whether the DNA was resuspended in 50 or 100 µl
aliquots. Gaining an appreciation of the numbers of cmuA-containing organisms
present in marine systems could allow estimations of the proportion of marine CH3Br
degradation that these organisms are responsible for. It is not known how sensitive that
the cmuA PCR is in terms of numbers of gene copies capable of being detected. It
seems from these data that, independent of the limit of detection of this PCR reaction,
the cmuA containing bacteria are a small fraction of total bacterial biomass. What these
data cannot show is how active the cmuA-containing bacteria are in consumption of
marine CH3Br, so although the numbers of these organisms present may be low, their
contribution to CH3Br degradation could be proportionally greater than that of other
bacterial communities involved in this process.
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Chapter 7
Leisingera methylohalidivorans strain
MB2 and attempts to identify cmuA
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7 Leisingera methylohalidivorans MB2 and Attempts to Identify cmuA
7.1 Introduction
The cmuA gene successfully PCR amplified from marine DNA samples (Chapter 6
and Schaefer et al., 2005); however cmuA has not been amplified from the single
marine isolate capable of growth on CH3Br as sole source of carbon and energy that
was available at the start of this study, Leisingera methylohalidivorans MB2. This
strain was isolated by Dr. Kelly Goodwin from a tidal pool in California (Schaefer et
al., 2002) and was capable of growth on MeBr as sole source of carbon and energy.
Properties of this organism are described in Chapter 1, section 1.4.4.
Attempts to amplify cmuA PCR products from this organism had repeatedly failed
using the primers cmuAF802 and cmuAR1609. Southern hybridisation, using probes
based on random priming of cmuA PCR products amplified from Aminobacter
ciceronei IMB-1, Aminobacter lissarensis CC495, Methylobacterium
chloromethanicum CM4 and Hyphomicrobium chloromethanicum CM2 also failed to
identify a cmuA in L. methylohalidivorans MB2. This indicates that the organism
either possesses a CmuA significantly different to those of the terrestrial strains or
that the gene was not present (Warner, 2003).
SDS PAGE of cell-free extracts of strain MB2 grown with and without CH3Br on
Marine Broth 2216 (Difco) were inconclusive, and there were no demonstrateable
differences between the polypeptide profiles of cells grown under the two growth
conditions, suggesting: (i) that cmuA was not expressed; (ii) that the inducible system
had ceased to be expressed; (iii) that cmuA associated protein in strain MB2, which
146
would be in contrast to previous findings (Schaefer et al., 2002); (iv) that cmuA was
not present in this organism.
Representatives of three further clades of MeBr-utilising bacteria that were distinct
from L. methylohalidivorans MB2, have recently been isolated (Schäfer et al, 2005).
The strains isolated (179, 198 and 217) were all identified as members of the
Roseobacter group of the α-Proteobacteria according to phylogenet. Strain 179
represents a potentially novel genus, most closely related to strains implicated in
juvenile oyster disease (Boettcher et al., 1999). Strain 198 clusters within the genus
Ruegeria and strain 217 is most closely related to the genus Roseovarius. The partial
cmu clusters from strains 179 and 198, but not from strain 217, were cloned and
sequenced (Schaefer et al., 2005). Alignments of the six available complete cmuA
sequences (strains CM4, CM2, IMB1, CC495, 179 and 198) with primers cmuAF802
and cmuAR1609 demonstrated significant mismatches with the cmuA sequences from
the marine isolates, particularly with the reverse primer and strain 198 whose cmuA
sequence could not be amplified with cmuAF802/ cmuAR1609 (see figs 7.1 and 7.2).
Fig 7.1. Alignment of cmuA sequences with primer cmuAF802, 5’-3’. Position numbering is based on that of M. chloromethanicum CM4. Bases complementary to the primer are shaded black and mismatches shaded grey.
147
Fig 7.2. Alignment of cmuA sequences with primer cmuAR1609. Position numbering is based on that of M. chloromethanicum CM4. The primer target site is shown alongside the reverse complement of the primer sequence. Shading as Fig 7.1.
Therefore a new reverse primer was designed based on all six complete sequences
together with the partial sequences available in the cmuA ARB database (~100
sequences) keeping both the number of redundancies and the number of mismatches
as low as possible. This primer was designed by Dr H. Schaefer (Fig 7.3).
Fig 7.3. Alignment of cmuA sequences with primer cmuAR1244. Position numbering is based on that of M. chloromethanicum CM4. The primer target site is shown alongside the reverse complement of the primer sequence. Shading as Fig 7.1.
With the new primer combination of cmuAF802/cmuAR1244, PCR products could
still not be amplified from L. methylohalidivorans MB2, but this primer provided the
basis for my attempt to obtain the cmu cluster from this organism.
7.2 Rationale and primer design
The corrinoid-binding domain of the bifunctional enzyme CmuA is the most highly
conserved region of the molecule. The structure of corrinoid-binding domains is
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known to be a four-helix bundle (Fig 7.4, Ludwig & Matthews, 1997) and structural
analysis of the C-terminal domain indicates that this is also the case for CmuA.
Fig 7.4. 3D-views of a four helix bundle corrinoid-binding domain with bound cobalamine. Top-down and end-on views of the binding niche are shown in a and b respectively. α-helices are shown as large pink arrows and cobalamine in ball-and-stick format. The bound cobalt of cobalamine is indicated Co. Structures downloaded from the Conserved Domain Database (see text) and redrawn with Cn3D 4.1 available from http://ncbi.nih.gov/Structure/CN3D/cn3d.shtml.
There are a number of motifs present that are conserved not only within bacterial
corrinoid-binding proteins, but also archaeal and eukaryotic, for example, the vitamin
B12 dependent human 5-methyl tetrahydrofolate-homocysteine methyltransferase
(Evans et al., 2002). BLASTp (Altschul et al., 1997) and the Conserved Domain
Database (Marchler-Bauer et al., 2005) search identifies the common motif MXXVG,
which is conserved as MKX V/I G in CmuA sequences obtained thus far. Craig
McAnulla proposed a second motif based on alignments with other corrinoid proteins
involved in methyl transfer, such as MetH, the cobalamin-dependent methionine
synthase of Escherichia coli (Old et al., 1990) and MtmC, a corrinoid protein
involved in methyl transfer from methanol to coenzyme M in Methanosarcina barkeri
a b
149
(LeClerc & Grahame, 1996; McAnulla, 2000). Within the proposed CmuA motif
NxQxxGx41SxMx28GG, the residues are believed to be involved in binding of the
corrinoid group, with the asparagine, glutamine and glycine residues forming a ligand
triad essential for enzyme activity in other corrinoid proteins (Ludwig & Matthews,
1997).
The corresponding protein sequence encoded by PCR primer cmuAR1244 is
positioned just inside the corrinoid-binding domain of cmuA. It is a feature of all the
primer pairs so far designed that they span both domains of this uniquely structured
enzyme, in order to increase specificity of the primers. This could also be responsible
for the lack of PCR product from L. methylohalidivorans. According to BLAST
searches, the most similar proteins are the mono- di- and tri-methylamine
methyltransferases and corrinoid-binding proteins of the methanogenic archaeon
Methanosarcina barkeri. These are present as separate enzymes in these organisms,
rather than the fusion of two functional domains, as with CmuA. It is possible that
this is also the case in L. methylohalidivorans and primers targeting only the
corrinoid-binding region might reveal whether this was the case. Primer cmuAR1352
(fig 7.5) was designed to amplify the corrinoid region when paired with the reverse
complement of cmuAR1244, cmuAF1224.
Fig 7.5. Alignment of cmuA sequences with primer cmuAR1352. Position numbering is based on that of M. chloromethanicum strain CM4. The primer target site is shown alongside the reverse complement of the primer sequence. Shading as Fig 7.1.
150
Primers were tested with A. ciceronei strain IMB-1 and H. chloromethanicum strain
CM2 and gave products of the expected size (128 bp) as demonstrated by gel
electrophoresis with ethidium bromide staining, running alongside Invitrogen 1kb
ladder. PCR conditions were as those in Chapter 2: Materials and Methods.
7.3 Results
A 128 bp PCR product was obtained using L. methylohalidivorans DNA as template.
This was cloned, sequenced and identified as cmuA by BLAST analysis, sharing 80 %
ID and with cmuA from A. lissarensis IMB1. Comparison was then made with the
rest of the cmuA database using ARB for phylogenetic analysis, which indicated that
the cmuA sequence from L. methylohalidivorans was different to those previously
sequenced. The cloned PCR product from L. methylohalidivorans was PCR amplified
again in order to gain enough of the PCR product for preparation of a gene probe for
Southern hybridisation analysis and to reveal the rest of the cmuA gene and cmu
cluster in this organism (probe preparation as Materials and Methods). Southern
analysis even at low washing stringencies (2 x SSC at 50 oC) failed to demonstrate
any hybridisation with the cmuA probe from L. methylohalidivorans, but the probe
bound extremely well to the positive control.
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Fig 7.6. Autoradiograph of L. methylohalidivorans Southern analysis. The orginal EtBr stained agarose gel image can be seen. Lanes 1, 8, 11 and 13 contain 1 kb DNA ladder (Invitrogen) and lane 14 contains the positive control, H. chloromethanicum cmuA in the Invitrogen TOPO vector. Lanes 2, 3, 4, 5, 6, and 7 contain L. methylohalidivorans DNA digested with, respectively restriction enzymes EcoRI, SalI, KpnI, BamHI, PstI and HindIII. Lanes 9, 10 and 12 contained Rhodobacteraceae strain 217 DNA digested with EcoRI, SalI and KpnI respectively. The blot was washed at a stringency of 2 x SSC at 50 oC and the X-ray film was exposed for 5 days with two enhancing screens. Arrowed are ladder bands (1636 bp) that have bound the probe.
On checking the sequence of the probe it was noticed that the base changes that
rendered the sequence novel were all at positions of degenerate bases in the primer
regions. Removing the primer sequences from the phylogenetic analysis indicated
that the PCR product obtained from the L. methylohalidivorans DNA was identical to
that of A. ciceronei, presumably due to low level contamination of the genomic DNA
sample.
1 2 3 4 5 6 7 8 9 1011121314
152
7.4 Discussion
The aim of this work was to test the hypothesis that L. methylohalidivorans MB2
possessed a cmuA substantially different from those previously identified and
sequenced. Despite the fact that they did not work in the case of L.
methylohalidivorans the primer pair developed in this work will prove useful for
obtaining cmuA sequences via Southern analysis from other organisms, as
demonstrated with H. chloromethanicum CM2 as the positive control for this analysis.
As the corrinoid-binding region is so well conserved in this enzyme, this region is
ideal for avoiding the mismatches present in other parts of the molecule.
Since this work was carried out, further attempts have been made to identify cmuA in
L. methylohalidivorans. A probe generated from a novel marine CH3Br utilising
bacterium, strain 179, was used in Southern hybridisation analysis and failed to obtain
hybridisation (Schaefer et al., 2005). During the course of this investigation it
became clear that Roseovarius strain 217, as with L. methylohalidivorans, did not
possess a cmuA identifiable by PCR. It also did not contain a distinct 67 kDa protein
present in SDS PAGE analysis when grown on CH3Br. It is therefore believed that
another pathway for the utilisation of methyl halides must be operating in these
organisms. Strain 217 has been accepted for genome sequencing by the Gordon and
Betty Moore Foundation and it is anticipated that this will provide some insight into
the mechanisms involved in these non-CmuA CH3X utilisers. It would be facile to
screen a genome sequence for methyltransferases and corrinoid-binding proteins, or
perhaps for other genes in the cmu cluster, such as cmuB, cmuC or folD
153
Chapter 8
Synopsis, Discussion, and Future Work
154
8 Chapter 8: Synopsis, Discussion and Future Work
8.1 Synopsis
8.1.1 Aims
The aims of this work were to investigate marine CH3Br-utilising bacteria and can be
split into four areas:
• Measurement of environmental (pptv) concentrations of CH3Br in seawater.
• Enrichment, isolation, and characterisation of CH3Br-utilising bacteria.
• Use of molecular ecological analyses to determine the presence, distribution
and diversity of CH3Br-utilising bacteria in seawater.
• Correlation of the presence and concentration of CH3Br with the presence and
abundance of CH3Br-utilising bacteria in seawater.
Two main sampling areas were used, the Arabian Sea through the NERC Marine and
Freshwater Microbial Biodiversity thematic AMBITION cruise, and L4, a sampling
station off the Coast of Plymouth (UK). A range of approaches was used, and these
are summarised below.
8.1.2 GC analysis of CH3Br
Three different gas chromatographic systems were used in order to measure CH3Br
concentrations: a GC FID to determine the presence/absence of CH3Br in enrichment
and other cultures; a GC ECD purge and trap system for extraction and analysis of
pptv concentrations of CH3Br from seawater samples; a second GC ECD purge and
trap system. The first GC ECD system suffered from electronic problems and was
superseded by the second system, which was used successfully to gather
measurements of seawater CH3Br concentrations from L4 over part of a seasonal
155
cycle. Rapid changes in concentrations of CH3Br from supersaturated to
undersaturated with respect to atmosphere. This suggested that the compound was
being degraded quickly, presumably by biological activity as this was more rapid than
could be accounted for by chemical degradation rates. The likelihood of in situ
production of CH3Br was also high further enhancing this fact. Peaks in CH3Br
supersaturations seemed to correspond with peaks in phytoplankton abundance, which
would be in agreement with data indicating the phytoplankton are an important
marine source of CH3Br (Baker et al., 1999; Saemundsdottir & Matrai, 1998; Scarratt
& Moore, 1998; Wingenter et al., 2004).
8.1.3 Enrichment and isolation
A large array of enrichments on CH3Br, CH3Cl and combinations of other C1
compounds was set up during the AMBITION cruise. Enrichments which contained
both CH3Br and 10 mM formate were able to be maintained on CH3Br for a long
time, whereas other enrichments from Arabian seawater were less successful.
Isolation of CH3Br-utilising bacteria was attempted from the active enrichments, and
although a number of strains were isolated, including one that gave a cmuA PCR
product, none were able to oxidise CH3Br in pure culture. Further active enrichments
were obtained from L4 seawater. Non-axenic cultures of the CH3Br-producing
coccolithophore Emiliania huxleyi CCMP 1516 were investigated as a potential
source of CH3Br-utilising isolates; this was unsuccessful.
8.1.4 MxaF diversity
PCR primers for the analysis of methanol dehydrogenase large subunit (MxaF)
sequences as a molecular marker of marine methylotrophic diversity were re-
developed. A number of potential PCR primers for mxaF were designed to target a
156
wider diversity of Proteobacterial mxaF sequences. Full-length sequences of α-, β-,
and γ-Proteobacterial mxaF sequences were aligned in order to enable this. These
mxaF PCR primers were screened, resulting in the new reverse primer mxaR1555,
which proved, in conjunction with the original forward primer mxaF1003, to be able
to amplify mxaF sequences from a large number of methanol-oxidising bacteria.
Although marine environmental samples were not tested within the timescale of this
work, the mxaF PCR primers show promise for use in future studies of both
methylotroph and methanotroph diversity.
8.1.5 cmuA diversity
The diversity of cmuA sequences in the active enrichments from both the Arabian Sea
and L4 was assessed by clone library analysis. Sequences belonged to three clades,
one novel clade with no cultured representatives and two clades not previously
containing any marine sequences.
Clone libraries were also produced from cmuA PCR products amplified from DNA of
large volume seawater samples from the Arabian Sea. Phylogenetic analysis of these
cmuA sequences indicated a shift in the most common sequence types observed in
these libraries that occurs between stations one and four of the cruise. These
sequences, together with available cmuA sequences from other environments, formed
an extensive database which was used to develop Terminal Restriction Fragment
Length Polymorphism analysis as a rapid technique for screening cmuA diversity in
the large number of remaining DNA samples from the Arabian Sea cruise, and
samples from a Celtic Sea cruise. Although the TRFLP technique was successfully
applied to standard clones, no cmuA PCR products were obtained from any of the
remaining samples. This was related to the sample volume used, with cmuA products
157
easily obtained from CH3Br enrichments and large volume samples, but not from
those with sample volumes of 2 L or less. This presumably reflects the abundance of
cmuA containing organisms in the marine environment at the time of sampling.
8.1.6 Leisingera methylohalidivorans MB2
L. methylohalidivorans was the single marine isolate capable of utilising CH3Br as
sole carbon and energy source available at the beginning of this study. Although a
number of attempts had been made to reveal the presence of the CmuA pathway in
this organism, none had been successful. It was hypothesized that L.
methylohalidivorans could contain a cmuA sequence substantially different to any
other previously identified. PCR primers were designed to the highly conserved
region encoding the corrinoid-binding region of cmuA. A 128 bp PCR product was
obtained from L. methylohalidivorans MB2 and used as a probe in Southern analysis.
This failed to hybridise and on sequencing of the probe, it was discovered that
ambiguities in the primer regions of the sequence had resulted in it being mistakenly
identified as novel, whereas in fact it was 100 % identical to the sequence of
Aminobacter ciceronei IMB1, and therefore due to contamination of the PCR.
8.2 Discussion and future work
The global CH3Br cycle has yet to be fully defined. The oceans are known to be both
a source and a sink of CH3Br, but the part that CH3Br-utilising bacteria play in this
marine flux of CH3Br remains obscure. Changes in diversity of the populations
responsible for CH3Br could have repercussions for the global cycle of this compound
and it is therefore important to be able to characterise these populations and their
involvement, not only to clarify current fluxes, but also to be able to model changes in
158
the degradation rates of CH3Br with respect to changes in bacterial community
structure.
The ultimate aim of this investigation was to couple measurements of the fluxes of
CH3Br with molecular microbiological methods in order to characterise the
contribution of CH3Br-utilising bacteria to the marine CH3Br sink. This was an
ambitious aim and although not achieved, this investigation has laid firm groundwork
for further work towards this goal.
8.2.1 Measurement of CH3Br
Development of GC techniques sensitive enough to detect environmental
concentrations of CH3Br (levels of pmol/l) allowed measurements of water column
CH3Br at station L4, off the coast of Plymouth, UK. Results indicated that levels of
CH3Br rapidly switched from supersaturated to undersaturated concentrations in
seawater, perhaps reflecting the activity of CH3Br-utilising bacteria. In order to
investigate this further, there are a number of experiments that could be undertaken.
GC ECD ‘system three’ with purge and trap apparatus could be used to measure rates
of loss of CH3Br when spiked into seawater samples at environmental concentrations.
Inclusion of inhibitors such as, mercuric chloride (to measure chemical loss rates) and
acetylene or methyl tert-butyl ether (to inhibit soluble methane monooxygenase,
which is capable of co-oxidation of CH3X) would allow assignment of the total loss
rates of CH3Br to various components of the bacterioplankton. It would be extremely
useful to develop an inhibitor of CmuA for use in this experiment in order to reveal
the amount of CH3Br degradation that this pathway is responsible for. (Hoeft et al.,
2000) made use of 500 µM chloroform as an inhibitor of transmethylation,
successfully inhibiting CH3Br consumption by strain LIS-3. It should be noted that
159
this concentration of chloroform is much higher than the concentration of CH3Br that
would be present and may have other, undesirable, effects on the natural population
present in incubations. Recently (Goodwin et al., 2005) demonstrated the inhibition
of CH3Br degradation in seawater samples by 160 to 200 nM toluene and isolated a
strain Oxy6 which was capable of growth on toluene whilst co-oxidising CH3Br.
Extension of the measurement of CH3Br concentrations at station L4 in coastal waters
off Plymouth over a full seasonal cycle would allow the determination of any seasonal
effects on CH3Br fluxes. CH3Br could also be measured during diel sampling cycles,
and over more detailed depth profiles. Critical to understanding the importance of
CmuA containing bacteria to degradation of marine CH3Br would be determination of
the number of these organisms present and their activity. The successful
amplification of cmuA from large volume DNA samples and enrichments gave an
appreciation of the low numbers of these organisms present, but development of Real
Time PCR for quantitative detection of cmuA copy number would be a major
objective. This technique has already been applied successfully to detection of
functional genes of methylotrophic bacteria such as pmoA in soils (Kolb et al., 2003).
Coupling this with RNA extraction and Reverse Transcriptase PCR would allow for
enumeration of cmuA that is actively being expressed. This would be a challenging
project to attempt given the indications of low levels of cmuA present in seawater
DNA extracts.
8.2.2 Molecular techniques
Two separate attempts were made to elucidate the diversity of CH3Br-utilising marine
bacteria by molecular methods. Firstly, the primer pair cmuAF802/cmuAR1609 was
applied to marine enrichment and DNA samples from Arabian Sea and Plymouth
160
coastal seawater and a large number of marine cmuA sequences were collected.
Falling into three clades, these sequences indicated a diversity that is not currently
represented by isolated CH3Br-utilising bacteria. There is certainly more scope for
future attempts at isolating these organisms, perhaps using a wider range of media, as
only one was used in this case. Two sequences belonging to clade A3 from the
pooled Arabian Sea enrichment PE2 contained in-frame stop codons, confirmed by
resequencing. It is possible that they could have been due to PCR or cloning errors,
but also possible that they were present in an organism in this condition. It is unclear
as to whether these genes would have been expressed in the environment. A future
study could make use of the RNA Stable-Isotope Probing (Manefield et al., 2002)) in
order to gain an impression of which clades are actively responsible for 13CH3Br-
utilisation, and to use DNA Stable-Isotope-Probing in order to look at cmuA diversity
in the active portion of the population. It would be critical to identify samples in
which CH3Br is being rapidly utilised prior to use of these stable-isotope techniques
as they rely on swift utilisation of heavy isotope labelled substrates in order that the
labelled carbon does not leach into the non-CH3Br utilising microbial assemblage via
consumption of secondary metabolites, cross-feeding, or other means. There is the
added complication when using seawater samples of chemical degradation of the
labelled CH3Br to methanol and CH3Cl via hydrolysis and nucleophilic substitution
reactions, which would also serve to label non-CH3X degrading DNA/RNA.
Coupling DNA/RNA SIP with metagenomic analyses such as fosmid or BAC library
production might allow the isolation of complete cmu clusters from only that portion
of the population that was actively utilising CH3Br and may also allow identification
of non-cmu pathway degradation mechanisms. Co-localisation of phylogenetic
marker genes with functional genes can allow the identification of the organism
161
responsible. Use of these simple, but powerful techniques has previously allowed the
identification of a completely novel form of bacterial phototrophy (Beja et al., 2000).
Secondly, mxaF, the gene encoding the large subunit of methanol dehydrogenase had
PCR primers re-developed with the aim of looking at the diversity of these sequences
within marine systems. Some, but not all, CH3X-utilising bacteria (including H.
chloromethanicum CM2 and M. chloromethanicum CM4) possess a methanol
dehydrogenase and this gene could be used as a phylogenetic marker to gain insights
into the phylogeny of a subset of CH3X-utilisers.
8.2.3 CH3Br-utilising isolates and enrichments
There are a number of indications that bacteria utilising the cmu pathway are not the
only marine bacteria capable of degrading CH3Br. Methane oxidisers (Dalton &
Stirling, 1982; Stirling & Dalton, 1980), ammonia oxidisers (Rasche et al., 1990), and
propane oxidisers (Streger et al., 1999) have also been demonstrated to co-oxidise
CH3Br and are likely to contribute to marine degradation when present in this
environment.
Various attempts to demonstrate the presence of cmuA in Leisingera
methylohalidivorans MB2, including this one, have failed, although a primer for the
corrinoid-binding domain of cmuA with potential useful future applications, such as
amplification of cmuA sequences beyond the scope of the current primer set, was
designed in the process. The reason could simply be that a different pathway is being
employed in this organism to allow growth on CH3Br as sole carbon and energy
source. This pathway could be identified by techniques such as transposon
mutagenesis and the isolation of mutants unable to utilise CH3Br as sole carbon and
162
energy source. However, this would rely on the development of genetic techniques
for this organism, and attempts would be further hampered by its low biomass
production when grown on CH3Br.
During this project Schaefer et al., 2005 isolated 13 novel marine CH3Br-utilising
bacteria belonging to three clades of α-Proteobacteria. Two of these clades,
represented by Rhodobacteracaea strains 179 and 198, were demonstrated by SDS
PAGE and protein mass spectrometry analysis to express CmuA when grown on
CH3Br and cmu clusters were subsequently cloned and sequenced from these.
However, the third clade, represented by Rhodobacteracaea strain 217, did not. This
strain was also capable of utilising dimethyl sulfoniopropionate (DMSP) as sole
carbon and energy source its genome was recently sequenced through funding by the
Gordon and Betty Moore Foundation. Currently the genome is awaiting annotation.
After analysis, it might be possible to identify a candidate pathway for CH3Br-
utilisation, and this could be used to identify the CH3X-degrading system in L.
methylohalidivorans MB2.
Goodwin et al., 2005 tested the ability of toluene to inhibit CH3Br consumption by L.
methylohalidivorans MB2 and found that it did not. Toluene also did not inhibit
CH3Br consumption by the cmu pathway organism Aminobacter ciceronei IMB1. As
toluene inhibited CH3Br degradation by 29-100 % in samples from the Western and
North Atlantic, North Pacific and Southern Ocean, but failed to inhibit either of these
organisms, it would be interesting to find out the proportion of CH3Br degradation
that the pathways represented by these two organisms contribute to total marine
CH3Br degradation. The toluene and CH3Br co-oxidising strain Oxy6 isolated by
163
these investigators was placed phylogenetically close to Erythrobacter and
Porphyrobacter clusters of the Sphingomonadacaea. One of the isolates from
Arabian Sea enrichments, MJC8 was identified by 16S rRNA sequencing to be related
to the Erythrobacter genus, and although it did not demonstrate the presence of cmuA
after PCR amplification, perhaps it was co-oxidising CH3Br in a similar manner to
strain Oxy6.
What is not yet clear from any analysis carried out to date is whether CH3X are used
by marine bacteria capable of their utilisation as sole carbon and energy source, or
whether these organisms mainly make use of other available growth substrates. In
this case degradation of CH3Br might be as a top-up energy source or top-up carbon
source, but not directly support biomass production. Thermodynamically CH3Br is a
viable energy source and it has been calculated that dissolved substrates are useful
energy sources even at the vanishingly low concentrations that CH3Br is present at in
marine systems (Williams, 2000). Expression of the cmu pathway enzymes in H.
chloromethanicum CM2 was tightly regulated in response to the presence of CH3Cl
and CH3Br (Borodina et al., 2004), even when the organism was being grown on an
alternative substrate. Further work needs to be carried to investigate the importance
of CH3X as a carbon and energy source in by CH3X-utilising bacteria.
During the enrichment and isolation of CH3Br-utilising bacteria, it was noticed that
the most active enrichments from the Arabian Sea were all enriched with CH3Br
together with 10 mM formate. The enzyme formate dehydrogenase (FDH) which
catalyses the oxidation of formate to carbon dioxide is part of the cmu CH3Br
164
utilisation pathway and perhaps enrichment with formate serendipitously enriched for
this pathway.
8.3 In conclusion
During this study, a sensitive GC ECD system for measurement of seawater CH3Br
concentrations was developed, CH3Br oxidising enrichments from contrasting marine
provinces were obtained and isolation of novel CH3Br-utilising bacteria was
attempted. Primers targeting the gene encoding for the large subunit of methanol
dehydrogenase were redesigned and shown to be useful for amplifying mxaF from a
wide range of methylotrophs and methanotrophs. A wide range of different cmuA
sequences was produced by clone library analysis from both Plymouth coastal, and
Arabian Sea seawater CH3Br enrichments and samples. These were used to develop
the TRFLP technique for use with cmuA and allow the rapid screening of these
sequences in environmental DNA samples in future.
Of the aims of this work, measurements of CH3Br in seawater were made and the
presence, distribution and diversity of the cmu pathway containing CH3Br-utilising
bacteria was assessed. Marine CH3Br enrichments were successfully produced,
although novel isolates were not obtained. With more time the critical aim would be
to couple measurements of CH3Br with the molecular techniques developed here.
164
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Appendices
181
A AMBITION Data
A.1 Data source
Data was obtained from the Biological Oceanography Data Centre, Liverpool, which
collated the data from RRS Charles Darwin cruise CD132 participants. The data is
to be made generally available in CD format. Data is credited to each of the cruise
participants involved below and the cruise report and data CD should be consulted
for details of methods. As there were several casts per station it was necessary to
select one per station for the sake of graphical representation. The most consistent
cast was referred to as the biogeochemistry cast and was carried out at dawn on every
station; data from this cast was used to produce the graphs below unless otherwise
stated. Station and cast numbers are in Table A.1 below.
Station Cast Location, Lat./Long. 1 1 00o54.3’S 64o08.5’E 2 1 00o00.9’S 67o00.0’E 3 9 03o47.8’N 67o00.0’E 4 6 07o36.0’N 67o00.0’E 5 6 11o24.0’N 67o00.0’E 6 7 15o12.0’N 67o00.0’E 7 11 19o00.0’N 67o00.0’E 8 5 20o55.0’N 63o40.0’E 9 5 23o33.7’N 59o54.2’E 10 6 24o20.0’N 58o10.0’E 11 6 26o00.0’N 56o35.1’E Table A.1. Location of casts used for data analyses.
It is worth noting that certain measurements were not taken at either the beginning or
end of the cruise, mainly due to the nature of the equipment or methods involved that
require time to set up and pack away for transport to and from the UK. The depth of
the measurements made also varies from station to station. The maximum is usually
250-300 m although at the later stations this is considerably reduced reflecting the
shallowing water.
182
A.2 Physicochemical Data
183
184
A.3 Productivity
185
186
A.4 Microorganism abundance
187
B List of Samples from the AMBITION Cruise
B.1 List of DNA Samples
ID No.
Station no.
Date Location Cast Depth (m)
Filter type Note
1 1 03/09/01 00o54’S 64o08’E 01 5 Supor Surface 2 1 03/09/01 00o54’S 64o08’E 01 74 Supor DCM 3 1 03/09/01 00o54’S 64o08’E 01 1 Supor 4 1 03/09/01 00o54’S 64o08’E 01 10 Supor 5 1 03/09/01 00o54’S 64o08’E 01 25 Supor 6 1 03/09/01 00o54’S 64o08’E 01 50 Supor 7 1 03/09/01 00o54’S 64o08’E 01 60 Supor 8 1 03/09/01 00o54’S 64o08’E 01 100 Supor 9 1 04/09/01 00o54’S 64o08’E 10 75 Supor DCM 10 1 04/09/01 00o54’S 64o08’E 10 1 Supor 11 1 04/09/01 00o54’S 64o08’E 10 10 Supor 12 1 04/09/01 00o54’S 64o08’E 10 25 Supor 13 1 04/09/01 00o54’S 64o08’E 10 65 Supor 14 1 04/09/01 00o54’S 64o08’E 10 85 Supor 15 1 04/09/01 00o54’S 64o08’E 10 140 Supor 16 1 04/09/01 00o54’S 64o08’E 11 5 Supor Surface 17 2 05/09/01 00o01’S 67o00’E 01 5 Supor Surface 18 2 05/09/01 00o01’S 67o00’E 01 62 Supor DCM 19 2 05/09/01 00o01’S 67o00’E 01 1 Supor 20 2 05/09/01 00o01’S 67o00’E 01 10 Supor 21 2 05/09/01 00o01’S 67o00’E 01 25 Supor 22 2 05/09/01 00o01’S 67o00’E 01 50 Supor 23 2 05/09/01 00o01’S 67o00’E 01 80 Supor 24 2 05/09/01 00o01’S 67o00’E 01 100 Supor 25 2 06/09/01 00o01’S 67o00’E 10 1 Sterivex 26 2 06/09/01 00o01’S 67o00’E 10 5 Sterivex Surface 27 2 06/09/01 00o01’S 67o00’E 10 10 Sterivex 28 2 06/09/01 00o01’S 67o00’E 10 25 Sterivex 29 2 06/09/01 00o01’S 67o00’E 10 50 Sterivex DCM 30 2 06/09/01 00o01’S 67o00’E 10 60 Sterivex 31 2 06/09/01 00o01’S 67o00’E 10 100 Sterivex 32 2 06/09/01 00o01’S 67o00’E 10 150 Sterivex 33 3 07/09/01 03o48’N 67o00 E 01 1 Sterivex Diel 1 34 3 07/09/01 03o48’N 67o00 E 01 70 Sterivex Diel 1 35 3 07/09/01 03o48’N 67o00 E 02 1 Sterivex Diel 2 36 3 07/09/01 03o48’N 67o00 E 02 70 Sterivex Diel 2 37 3 08/09/01 03o48’N 67o00 E 04 1 Sterivex Diel 3 38 3 08/09/01 03o48’N 67o00 E 04 71 Sterivex Diel 3 39 3 08/09/01 03o48’N 67o00 E 05 1 Sterivex Diel 4 40 3 08/09/01 03o48’N 67o00 E 05 70 Sterivex Diel 4 41 3 09/09/01 03o48’N 67o00 E 09 5 Supor Surface
188
ID No.
Station No.
Date Location Cast Depth Filter type Note
42 3 09/09/01 03o48’N 67o00 E 09 63 Supor DCM 43 3 09/09/01 03o48’N 67o00 E 09 1 Supor 44 3 09/09/01 03o48’N 67o00 E 09 10 Supor 45 3 09/09/01 03o48’N 67o00 E 09 25 Supor 46 3 09/09/01 03o48’N 67o00 E 09 50 Supor 47 3 09/09/01 03o48’N 67o00 E 09 80 Supor 48 3 09/09/01 03o48’N 67o00 E 09 100 Supor 49 4 10/09/01 07o35’N 67o00 E 02 5/1 Sterivex Surface 50 4 10/09/01 07o35’N 67o00 E 02 66 Sterivex DCM 51 4 10/09/01 07o35’N 67o00 E 02 90 Sterivex 52 4 11/09/01 07o35’N 67o00 E 06 5 Supor Surface 53 4 11/09/01 07o35’N 67o00 E 06 77 Supor DCM 54 4 11/09/01 07o35’N 67o00 E 06 1 Supor 55 4 11/09/01 07o35’N 67o00 E 06 10 Supor 56 4 11/09/01 07o35’N 67o00 E 06 25 Supor 57 4 11/09/01 07o35’N 67o00 E 06 50 Supor 58 4 11/09/01 07o35’N 67o00 E 06 65 Supor 59 4 11/09/01 07o35’N 67o00 E 06 90 Supor 60 5 12/09/01 11o06’N 66o59 E 02 5 Supor 61 5 12/09/01 11o06’N 66o59 E 02 5 Supor 0.45 62 5 12/09/01 11o06’N 66o59 E 02 60 Supor 63 5 12/09/01 11o06’N 66o59 E 02 60 Supor 0.45 64 5 12/09/01 11o06’N 66o59 E 02 100 Supor 65 5 12/09/01 11o06’N 66o59 E 02 100 Supor 0.45 66 5 13/09/01 11o06’N 66o59 E 06 5 Supor Surface 67 5 13/09/01 11o06’N 66o59 E 06 36 Supor DCM 68 5 13/09/01 11o06’N 66o59 E 06 1 Supor 69 5 13/09/01 11o06’N 66o59 E 06 10 Supor 70 5 13/09/01 11o06’N 66o59 E 06 25 Supor 71 5 13/09/01 11o06’N 66o59 E 06 50 Supor 72 5 13/09/01 11o06’N 66o59 E 06 80 Supor 73 5 13/09/01 11o06’N 66o59 E 06 100 Supor 74 6 14/09/01 15o12’N 67o00 E 02 5 Sterivex Surface 75 6 14/09/01 15o12’N 67o00 E 02 35 Sterivex DCM 76 6 14/09/01 15o12’N 67o00 E 02 60 Sterivex 77 6 14/09/01 15o12’N 67o00 E 02 120 Sterivex 78 6 14/09/01 15o12’N 67o00 E 02 202 Sterivex 79 6 14/09/01 15o12’N 67o00 E 02 701 Sterivex 80 6 14/09/01 15o12’N 67o00 E 02 1600 Sterivex 81 6 14/09/01 15o12’N 67o00 E 02 2501 Sterivex 82 6 15/09/01 15o12’N 67o00 E 07 5 Supor Surface 83 6 15/09/01 15o12’N 67o00 E 07 40 Supor DCM 84 6 15/09/01 15o12’N 67o00 E 07 10 Supor 85 6 15/09/01 15o12’N 67o00 E 07 20 Supor 86 6 15/09/01 15o12’N 67o00 E 07 50 Supor 87 6 15/09/01 15o12’N 67o00 E 07 75 Supor
189
ID No.
Station No.
Date Location Cast Depth Filter type Note
88 7 16/09/01 19o00’N 67o00 E 03 5 Sterivex Diel 1 89 7 16/09/01 19o00’N 67o00 E 03 47 Sterivex Diel 1 90 7 16/09/01 19o00’N 67o00 E 04 5 Sterivex Diel 2 91 7 16/09/01 19o00’N 67o00 E 04 50 Sterivex Diel 2 92 7 17/09/01 19o00’N 67o00 E 05 5 Sterivex Diel 3 93 7 17/09/01 19o00’N 67o00 E 05 50 Sterivex Diel 3 94 7 17/09/01 19o00’N 67o00 E 06 5 Sterivex Diel 4 95 7 17/09/01 19o00’N 67o00 E 06 52 Sterivex Diel 4 96 7 17/09/01 19o00’N 67o00 E 07 5 Sterivex Diel 5 97 7 17/09/01 19o00’N 67o00 E 07 50 Sterivex Diel 5 98 7 18/09/01 19o00’N 67o00 E 11 5 Supor Surface 99 7 18/09/01 19o00’N 67o00 E 11 49 Supor DCM 100 7 18/09/01 19o00’N 67o00 E 11 10 Supor 101 7 18/09/01 19o00’N 67o00 E 11 20 Supor 102 7 18/09/01 19o00’N 67o00 E 11 40 Supor 103 7 18/09/01 19o00’N 67o00 E 11 60 Supor 104 7 18/09/01 19o00’N 67o00 E 11 80 Supor 105 7 18/09/01 19o00’N 67o00 E 11 100 Supor 106 8 19/09/01 20o55’N 63o40 E 02 5 Sterivex Surface 107 8 19/09/01 20o55’N 63o40 E 02 10 Sterivex 108 8 19/09/01 20o55’N 63o40 E 02 18 Sterivex 109 8 19/09/01 20o55’N 63o40 E 02 21 Sterivex DCM 110 8 19/09/01 20o55’N 63o40 E 02 40 Sterivex 111 8 19/09/01 20o55’N 63o40 E 02 60 Sterivex 112 8 19/09/01 20o55’N 63o40 E 02 150 Sterivex 113 8 19/09/01 20o55’N 63o40 E 02 250 Sterivex 114 8 20/09/01 20o55’N 63o40 E 05 5 Supor Surface 115 8 20/09/01 20o55’N 63o40 E 05 29 Supor DCM 116 8 20/09/01 20o55’N 63o40 E 05 250 Supor 117 8 20/09/01 20o55’N 63o40 E 05 10 Supor 118 8 20/09/01 20o55’N 63o40 E 05 20 Supor 119 8 20/09/01 20o55’N 63o40 E 05 50 Supor 120 8 20/09/01 20o55’N 63o40 E 05 70 Supor 121 8 20/09/01 20o55’N 63o40 E 05 100 Supor 122 9 22/09/01 23o33’N 59o54 E 02 5 Supor CM 123 9 22/09/01 23o33’N 59o54 E 02 10 Supor 124 9 22/09/01 23o33’N 59o54 E 02 20 Supor 125 9 22/09/01 23o33’N 59o54 E 02 40 Supor 126 9 22/09/01 23o33’N 59o54 E 02 100 Supor 127 9 22/09/01 23o33’N 59o54 E 02 210 Supor 128 9 22/09/01 23o33’N 59o54 E 02 230 Supor 129 9 22/09/01 23o33’N 59o54 E 02 250 Supor CM 130 9 23/09/01 23o33’N 59o54 E 05 2.5 Supor CM 131 9 23/09/01 23o33’N 59o54 E 05 7.5 Supor 132 9 23/09/01 23o33’N 59o54 E 05 10 Supor 133 9 23/09/01 23o33’N 59o54 E 05 20 Supor
190
ID No.
Station No.
Date Location Cast Depth Filter type Note
134 9 23/09/01 23o33’N 59o54 E 05 40 Supor 135 9 23/09/01 23o33’N 59o54 E 05 70 Supor 136 9 23/09/01 23o33’N 59o54 E 05 90 Supor 137 9 23/09/01 23o33’N 59o54 E 05 110 Supor 138 10 24/09/01 24o20’N 58o10 E 02 5 Supor 139 10 24/09/01 24o20’N 58o10 E 02 20 Supor 140 10 24/09/01 24o20’N 58o10 E 02 29 Supor DCM 141 10 24/09/01 24o20’N 58o10 E 02 55 Supor 142 10 24/09/01 24o20’N 58o10 E 02 60 Supor 143 10 24/09/01 24o20’N 58o10 E 02 70 Supor 144 10 24/09/01 24o20’N 58o10 E 02 100 Supor 145 10 24/09/01 24o20’N 58o10 E 02 120 Supor 146 10 25/09/01 24o20’N 58o10 E 06 5 Supor Surface 147 10 25/09/01 24o20’N 58o10 E 06 27 Supor DCM 148 10 25/09/01 24o20’N 58o10 E 06 15 Supor 149 10 25/09/01 24o20’N 58o10 E 06 20 Supor 150 10 25/09/01 24o20’N 58o10 E 06 36 Supor 151 10 25/09/01 24o20’N 58o10 E 06 55 Supor 152 10 25/09/01 24o20’N 58o10 E 06 65 Supor 153 10 25/09/01 24o20’N 58o10 E 06 90 Supor 154 11 26/09/01 26o00’N 56o35 E 02 5 Supor Surface 155 11 26/09/01 26o00’N 56o35 E 02 15 Supor 156 11 26/09/01 26o00’N 56o35 E 02 31 Supor CM 157 11 26/09/01 26o00’N 56o35 E 02 40 Supor 158 11 26/09/01 26o00’N 56o35 E 02 60 Supor 159 11 26/09/01 26o00’N 56o35 E 02 75 Supor 160 11 26/09/01 26o00’N 56o35 E 02 80 Supor 161 11 26/09/01 26o00’N 56o35 E 02 92 Supor Sal. max. 162 11 27/09/01 26o00’N 56o35 E 06 5 Supor Surface 163 11 27/09/01 26o00’N 56o35 E 06 26 Supor DCM 164 11 27/09/01 26o00’N 56o35 E 06 90 Supor Sal. max. 165 11 27/09/01 26o00’N 56o35 E 06 15 Supor 166 11 27/09/01 26o00’N 56o35 E 06 19 Supor 167 11 27/09/01 26o00’N 56o35 E 06 40 Supor 168 11 27/09/01 26o00’N 56o35 E 06 60 Supor 169 11 27/09/01 26o00’N 56o35 E 06 80 Supor Table B.1. List of AMBTION DNA samples. Notes are as follows: Surface, The sample was treated as the surface water sample, usually ~5 m; DCM, Deep Chlorophyll Maximum; CM, Chlorophyll Maximum; Sal. max., Salinity maximum, noted only in areas of high salinity; Diel, the sample was part of a diel sampling cycle; 0.45, Indicates the use of 0.45 µm filters to sample a different size fraction.
191
B.2 List of Enrichments Set-up On the AMBITION Cruise
ID No. Substrate Key Station Date Cast Depth (m) 1 1 1 03/09/01 1 5 2 2 1 03/09/01 1 5 3 3 1 03/09/01 1 5 4 4 1 03/09/01 1 5 5 5 1 03/09/01 1 5 6 6 1 03/09/01 1 5 7 7 1 03/09/01 1 5 8 8 1 03/09/01 1 5 9 9 1 03/09/01 1 5 10 10 1 03/09/01 1 5 11 11 1 03/09/01 1 5 12 12 1 03/09/01 1 5 13 1 1 03/09/01 1 74 14 2 1 03/09/01 1 74 15 3 1 03/09/01 1 74 16 4 1 03/09/01 1 74 17 5 1 03/09/01 1 74 18 6 1 03/09/01 1 74 19 7 1 03/09/01 1 74 20 8 1 03/09/01 1 74 21 9 1 03/09/01 1 74 22 10 1 03/09/01 1 74 23 11 1 03/09/01 1 74 24 12 1 03/09/01 1 74 25 1 2 05/09/01 1 5 26 2 2 05/09/01 1 5 27 3 2 05/09/01 1 5 28 4 2 05/09/01 1 5 29 5 2 05/09/01 1 5 30 6 2 05/09/01 1 5 31 7 2 05/09/01 1 5 32 8 2 05/09/01 1 5 33 9 2 05/09/01 1 5 34 10 2 05/09/01 1 5 35 11 2 05/09/01 1 5 36 12 2 05/09/01 1 5 37 1 2 05/09/01 1 62 38 2 2 05/09/01 1 62 39 3 2 05/09/01 1 62 40 4 2 05/09/01 1 62 41 5 2 05/09/01 1 62 42 6 2 05/09/01 1 62 43 7 2 05/09/01 1 62 44 8 2 05/09/01 1 62
192
ID No. Substrate Key Station Date Cast Depth (m) 45 9 2 05/09/01 1 62 46 10 2 05/09/01 1 62 47 11 2 05/09/01 1 62 48 12 2 05/09/01 1 62 49 1 3 09/09/01 9 5 50 2 3 09/09/01 9 5 51 3 3 09/09/01 9 5 52 4 3 09/09/01 9 5 53 5 3 09/09/01 9 5 54 6 3 09/09/01 9 5 55 7 3 09/09/01 9 5 56 8 3 09/09/01 9 5 57 9 3 09/09/01 9 5 58 10 3 09/09/01 9 5 59 11 3 09/09/01 9 5 60 12 3 09/09/01 9 5 61 1 3 09/09/01 9 63 62 2 3 09/09/01 9 63 63 3 3 09/09/01 9 63 64 4 3 09/09/01 9 63 65 5 3 09/09/01 9 63 66 6 3 09/09/01 9 63 67 7 3 09/09/01 9 63 68 8 3 09/09/01 9 63 69 9 3 09/09/01 9 63 70 10 3 09/09/01 9 63 71 11 3 09/09/01 9 63 72 12 3 09/09/01 9 63 73 1 4 11/09/01 6 5 74 2 4 11/09/01 6 5 75 3 4 11/09/01 6 5 76 4 4 11/09/01 6 5 77 5 4 11/09/01 6 5 78 6 4 11/09/01 6 5 79 7 4 11/09/01 6 5 80 8 4 11/09/01 6 5 81 9 4 11/09/01 6 5 82 10 4 11/09/01 6 5 83 11 4 11/09/01 6 5 84 12 4 11/09/01 6 5 85 1 4 11/09/01 6 77 86 2 4 11/09/01 6 77 87 3 4 11/09/01 6 77 88 4 4 11/09/01 6 77 89 5 4 11/09/01 6 77 90 6 4 11/09/01 6 77 91 7 4 11/09/01 6 77
193
ID No. Substrate Key Station Date Cast Depth (m) 92 8 4 11/09/01 6 77 93 9 4 11/09/01 6 77 94 10 4 11/09/01 6 77 95 11 4 11/09/01 6 77 96 12 4 11/09/01 6 77 97 1 5 13/09/01 6 5 98 2 5 13/09/01 6 5 99 3 5 13/09/01 6 5 100 4 5 13/09/01 6 5 101 5 5 13/09/01 6 5 102 6 5 13/09/01 6 5 103 7 5 13/09/01 6 5 104 8 5 13/09/01 6 5 105 9 5 13/09/01 6 5 106 10 5 13/09/01 6 5 107 11 5 13/09/01 6 5 108 12 5 13/09/01 6 5 109 1 5 13/09/01 6 36 110 2 5 13/09/01 6 36 111 3 5 13/09/01 6 36 112 4 5 13/09/01 6 36 113 5 5 13/09/01 6 36 114 6 5 13/09/01 6 36 115 7 5 13/09/01 6 36 116 8 5 13/09/01 6 36 117 9 5 13/09/01 6 36 118 10 5 13/09/01 6 36 119 11 5 13/09/01 6 36 120 12 5 13/09/01 6 36 121 1 6 14/09/01 2 2501 122 2 6 14/09/01 2 2501 123 3 6 14/09/01 2 2501 124 4 6 14/09/01 2 2501 125 5 6 14/09/01 2 2501 126 6 6 14/09/01 2 2501 127 7 6 14/09/01 2 2501 128 8 6 14/09/01 2 2501 129 9 6 14/09/01 2 2501 130 10 6 14/09/01 2 2501 131 11 6 14/09/01 2 2501 132 12 6 14/09/01 2 2501 133 1 6 15/09/01 7 5 134 2 6 15/09/01 7 5 135 3 6 15/09/01 7 5 136 4 6 15/09/01 7 5 137 5 6 15/09/01 7 5 138 6 6 15/09/01 7 5
194
ID No. Substrate Key Station Date Cast Depth (m) 139 7 6 15/09/01 7 5 140 8 6 15/09/01 7 5 141 9 6 15/09/01 7 5 142 10 6 15/09/01 7 5 143 11 6 15/09/01 7 5 144 12 6 15/09/01 7 5 145 1 6 15/09/01 7 40 146 2 6 15/09/01 7 40 147 3 6 15/09/01 7 40 148 4 6 15/09/01 7 40 149 5 6 15/09/01 7 40 150 6 6 15/09/01 7 40 151 7 6 15/09/01 7 40 152 8 6 15/09/01 7 40 153 9 6 15/09/01 7 40 154 10 6 15/09/01 7 40 155 11 6 15/09/01 7 40 156 12 6 15/09/01 7 40 157 1 7 18/09/01 11 5 158 2 7 18/09/01 11 5 159 3 7 18/09/01 11 5 160 4 7 18/09/01 11 5 161 5 7 18/09/01 11 5 162 6 7 18/09/01 11 5 163 7 7 18/09/01 11 5 164 8 7 18/09/01 11 5 165 9 7 18/09/01 11 5 166 10 7 18/09/01 11 5 167 11 7 18/09/01 11 5 168 12 7 18/09/01 11 5 169 1 7 18/09/01 11 49 170 2 7 18/09/01 11 49 171 3 7 18/09/01 11 49 172 4 7 18/09/01 11 49 173 5 7 18/09/01 11 49 174 6 7 18/09/01 11 49 175 7 7 18/09/01 11 49 176 8 7 18/09/01 11 49 177 9 7 18/09/01 11 49 178 10 7 18/09/01 11 49 179 11 7 18/09/01 11 49 180 12 7 18/09/01 11 49 181 1 8 20/09/01 5 5 182 2 8 20/09/01 5 5 183 3 8 20/09/01 5 5 184 4 8 20/09/01 5 5 185 5 8 20/09/01 5 5
195
ID No. Substrate Key Station Date Cast Depth (m) 186 6 8 20/09/01 5 5 187 7 8 20/09/01 5 5 188 8 8 20/09/01 5 5 189 9 8 20/09/01 5 5 190 10 8 20/09/01 5 5 191 11 8 20/09/01 5 5 192 12 8 20/09/01 5 5 193 1 8 20/09/01 5 29 194 2 8 20/09/01 5 29 195 3 8 20/09/01 5 29 196 4 8 20/09/01 5 29 197 5 8 20/09/01 5 29 198 6 8 20/09/01 5 29 199 7 8 20/09/01 5 29 200 8 8 20/09/01 5 29 201 9 8 20/09/01 5 29 202 10 8 20/09/01 5 29 203 11 8 20/09/01 5 29 204 12 8 20/09/01 5 29 205 1 8 20/09/01 5 250 206 2 8 20/09/01 5 250 207 3 8 20/09/01 5 250 208 4 8 20/09/01 5 250 209 5 8 20/09/01 5 250 210 6 8 20/09/01 5 250 211 7 8 20/09/01 5 250 212 8 8 20/09/01 5 250 213 9 8 20/09/01 5 250 214 10 8 20/09/01 5 250 215 11 8 20/09/01 5 250 216 12 8 20/09/01 5 250 217 1 9 23/09/01 5 2.5 218 2 9 23/09/01 5 2.5 219 3 9 23/09/01 5 2.5 220 4 9 23/09/01 5 2.5 221 5 9 23/09/01 5 2.5 222 6 9 23/09/01 5 2.5 223 7 9 23/09/01 5 2.5 224 8 9 23/09/01 5 2.5 225 9 9 23/09/01 5 2.5 226 10 9 23/09/01 5 2.5 227 11 9 23/09/01 5 2.5 228 12 9 23/09/01 5 2.5 229 1 9 23/09/01 5 7.5 230 2 9 23/09/01 5 7.5 231 3 9 23/09/01 5 7.5 232 4 9 23/09/01 5 7.5
196
ID No. Substrate Key Station Date Cast Depth (m) 233 5 9 23/09/01 5 7.5 234 6 9 23/09/01 5 7.5 235 7 9 23/09/01 5 7.5 236 8 9 23/09/01 5 7.5 237 9 9 23/09/01 5 7.5 238 10 9 23/09/01 5 7.5 239 11 9 23/09/01 5 7.5 240 12 9 23/09/01 5 7.5 241 1 10 25/09/01 6 5 242 2 10 25/09/01 6 5 243 3 10 25/09/01 6 5 244 4 10 25/09/01 6 5 245 5 10 25/09/01 6 5 246 6 10 25/09/01 6 5 247 7 10 25/09/01 6 5 248 8 10 25/09/01 6 5 249 9 10 25/09/01 6 5 250 10 10 25/09/01 6 5 251 11 10 25/09/01 6 5 252 12 10 25/09/01 6 5 253 1 10 25/09/01 6 27 254 2 10 25/09/01 6 27 255 3 10 25/09/01 6 27 256 4 10 25/09/01 6 27 257 5 10 25/09/01 6 27 258 6 10 25/09/01 6 27 259 7 10 25/09/01 6 27 260 8 10 25/09/01 6 27 261 9 10 25/09/01 6 27 262 10 10 25/09/01 6 27 263 11 10 25/09/01 6 27 264 12 10 25/09/01 6 27 265 1 11 27/09/01 6 5 266 2 11 27/09/01 6 5 267 3 11 27/09/01 6 5 268 4 11 27/09/01 6 5 269 5 11 27/09/01 6 5 270 6 11 27/09/01 6 5 271 7 11 27/09/01 6 5 272 8 11 27/09/01 6 5 273 9 11 27/09/01 6 5 274 10 11 27/09/01 6 5 275 11 11 27/09/01 6 5 276 12 11 27/09/01 6 5 277 1 11 27/09/01 6 26 278 2 11 27/09/01 6 26 279 3 11 27/09/01 6 26
197
ID No. Substrate Key Station Date Cast Depth (m) 280 4 11 27/09/01 6 26 281 5 11 27/09/01 6 26 282 6 11 27/09/01 6 26 283 7 11 27/09/01 6 26 284 8 11 27/09/01 6 26 285 9 11 27/09/01 6 26 286 10 11 27/09/01 6 26 287 11 11 27/09/01 6 26 288 12 11 27/09/01 6 26 289 1 11 27/09/01 6 90 290 2 11 27/09/01 6 90 291 3 11 27/09/01 6 90 292 4 11 27/09/01 6 90 293 5 11 27/09/01 6 90 294 6 11 27/09/01 6 90 295 7 11 27/09/01 6 90 296 8 11 27/09/01 6 90 297 9 11 27/09/01 6 90 298 10 11 27/09/01 6 90 299 11 11 27/09/01 6 90 300 12 11 27/09/01 6 90 Table B.2. List of AMBTION enrichments. Conditions refer to those in Table 2.3 of the Materials and methods.
198
C Henry’s Law
Throughout this thesis calculations of the concentrations of gases added to gas-tight
vials were made using Henry’s Law based on the dimensionless Henry’s Law
constants of MeBr, MeCl and methane. Henry’s law constants very with temperature
and are determined empirically and therefore can only be an approximation of the
actual concentration. They also assume that the gas in the headspace of a vial and
the gas in the aqueous phase are at equilibrium, which is not necessarily the case
with consumption or production of the gases, and that the liquid phase is pure water
rather than media as in this case.
Henry’s law constants for CH3Br and CH3Cl were calculated for the particular
temperature required using the Henry’s Law calculator available at the following
URL: http://www.epa.gov/athens/learn2model/part-two/onsite/esthenry.htm. The
constant for methane was obtained from Kim et al., 1999, quoting Yaws et al., 1991.
It was only available for 25 oC and this should be borne in mind when incubation
temperatures differ from this value.
An Excel spreadsheet was set up which had inputs (in yellow in Figure C.1) of the
dimensionless Henry’s Law constant at the relevant temperature, the volume of
headspace (mL), the volume of media (mL), the headspace concentration (% vol/vol)
and the temperature (oC). In the example below the media concentration of a 1L vial
containing 300 mL media at 20 oC is calculated for 0.2 % (vol/vol) MeBr. The
section to the right of the table not in bold contains the sub-calculations required
prior to the final one. The gas constant is temperature dependant and therefore
recalculated for each Henry’s Law calculation based on the equation P V = n R T,
199
where P is atmospheric pressure in N m-2 (101325 N m-2), V is the volume, n is the
number of moles (in this case 1), R is the universal gas constant, 8.314 when using N
m-2 as units of pressure, and T is temperature in oK.
Fig. C.1. Excel spreadsheet for Henry’s Law calculations
Henry's Law Constant (at relevant temp)
0.221 Headspace times Henry 154.7
Initial Headspace % 0.2 Above times media volume
454.7
Headspace volume 700 Headspace % times headspace volume
140
Volume of medium 300 Temp. in Kelvin 293.15 Temp in degrees C 20 Gas constant 24.05377844 % Total gas in medium 0.307895316 Concentration of gas in media in µM
128.00289
200
D TRF assignment of cmuA sequences
Sequence Clade HaeII F TRF
BsiYI F TRF
BsiYI R TRF
HpaII R TRF
Total
AY934476 A1 167 Uncut Uncut 156 323 AY934475 A1 167 Uncut Uncut 156 323 AY934471 A1 167 Uncut Uncut 156 323 AY934470 A1 167 Uncut Uncut 156 323 AY934459 A1 167 Uncut Uncut 156 323 AY934469 A1 167 Uncut Uncut 156 323 AY934462 A1 167 Uncut Uncut 156 323 H. chloromethanicum CM2
A1 167 Uncut Uncut 156 323
AY439205 A1 167 Uncut Uncut 156 323 Hyphomicrobium sp. 30 A1 167 Uncut Uncut 156 323 Hyphomicrobium sp. S4 ND 167 Uncut Uncut 156 323 AY934477 A1 167 Uncut Uncut 156 323 AY934454 A1 167 Uncut Uncut 156 323 AY934462 A1 167 Uncut Uncut 156 323 AY934472 A1 167 Uncut Uncut 156 323 AY934434 A4 145 Uncut Uncut 225 370 AY934448 A4 308 Uncut Uncut 156 464 Hyphomicrobium sp. LAT3
A4 53 Uncut Uncut 481 534
M. chloromethanicum CM4
Root 479 Uncut Uncut 110 589
AY439210 B3 34 126 325 135 620 AY439209 B3 34 126 325 135 620 AY439207 B3 34 126 325 135 620 AY934427 B3 34 126 325 135 620 AY439203 B3 34 126 325 135 620 AY934447 B3 34 126 325 135 620 AY934437 B3 34 126 325 135 620 AY934445 B3 34 126 325 135 620 AY934443 B3 34 126 325 135 620 A. lissarensis CC495 B3 34 126 325 135 620 AY934438 A4 145 Uncut Uncut 483 628 AY934430 A4 145 Uncut Uncut 483 628 AY439211 B3 43 126 325 135 629 DQ090684 B2 34 384 114 115 647 AY934474 B3 34 126 166 459 785 AY934473 A1 167 Uncut Uncut 625 792 DQ090704 A3 145 276 347 90 858 DQ090705 B1 53 329 171 330 883 AJ810831 B4 121 467 172 135 895 AJ810833 B4 121 467 172 135 895 AJ810832 B4 121 467 172 135 895 AY439208 B2 53 384 335 135 907
201
AY439206 B2 53 384 335 135 907 AY439201 B2 53 384 335 135 907 DQ090675 A3 145 276 347 144 912 DQ090668 A3 145 276 347 144 912 DQ090677 A3 145 276 347 144 912 DQ090669 A3 145 276 347 144 912 DQ090683 A3 145 276 347 144 912 DQ090667 A3 145 276 347 144 912 DQ090672 A3 145 276 347 144 912 DQ090680 A3 145 276 347 144 912 DQ090699 A3 145 276 347 144 912 DQ090665 A3 145 276 347 144 912 DQ090666 A3 145 276 347 144 912 DQ090676 A3 145 276 347 144 912 DQ090671 A3 145 276 347 144 912 DQ090670 A3 145 276 347 144 912 DQ090674 A3 145 276 347 144 912 DQ090673 A3 145 276 347 144 912 DQ090697 A3 145 276 347 144 912 DQ090679 A3 145 276 347 144 912 DQ090698 A3 145 276 347 144 912 Rhodobacteracaea 179 B1 43 402 347 135 927 AY934461 B1 43 402 347 135 927 AY934456 B1 43 402 347 135 927 AY439204 B1 34 50 764 135 983 AY934467 A2 121 461 347 135 1064 AY934466 A2 121 461 347 135 1064 AY934464 A2 121 461 347 135 1064 AY934463 A2 121 461 347 135 1064 AJ810828 A1 121 461 347 135 1064 AJ810829 A1 121 461 347 135 1064 AJ810830 A1 121 461 347 135 1064 AY934460 A2 121 461 347 135 1064 DQ090681 A3 308 276 347 144 1075 AY934457 A2 121 461 347 156 1085 AY934458 A2 121 461 347 156 1085 DQ090689 B1 53 329 391 330 1103 DQ090686 B1 53 329 391 330 1103 DQ090702 B1 53 329 391 330 1103 DQ090690 B1 53 329 391 330 1103 DQ090687 B1 53 329 391 330 1103 DQ090685 B1 53 329 391 330 1103 DQ090703 B1 53 329 391 330 1103 DQ090701 B1 53 329 391 330 1103 DQ090700 B1 53 329 391 330 1103 AY934440 A4 145 483 166 483 1277 AY934446 A4 145 329 347 459 1280 DQ090694 B2 34 384 406 483 1307
202
DQ090678 B2 34 384 406 483 1307 DQ090693 B2 34 384 406 483 1307 DQ090691 B2 34 384 406 483 1307 DQ090696 B2 34 384 406 483 1307 DQ090692 B2 34 384 406 483 1307 DQ090695 B2 34 384 406 483 1307 A. ciceronei IMB-1 B2 34 384 406 483 1307 DQ090688 B2 34 384 406 483 1307 AY934436 A4 145 346 347 483 1321 AY934441 A4 145 464 282 483 1374 AY439212 B1 271 397 417 330 1415 AY439202 B1 271 397 417 330 1415 AY439200 B1 271 397 417 330 1415 AY934442 A4 145 384 406 483 1418 Hyphomicrobium sp. SAC1
ND 167 384 427 459 1437
AY934453 A4 145 464 347 483 1439 AY934429 A4 145 464 347 483 1439 AY934478 A4 145 464 347 483 1439 AY934454 A4 145 464 347 483 1439 AY934435 A4 145 464 347 483 1439 AY934435 A4 145 464 347 483 1439 AY934433 A4 145 464 347 483 1439 AY934481 A4 145 464 347 483 1439 AY934426 A4 145 464 347 483 1439 AY934428 A4 145 464 347 483 1439 AY934442 A4 145 464 347 483 1439 AY934480 A4 145 464 347 483 1439 AY934479 A4 145 464 347 483 1439 AY934444 A4 145 464 347 483 1439 AY934432 A4 145 464 347 483 1439 AY934439 A4 145 464 347 483 1439 AY934450 A4 145 464 347 483 1439 AY934448 A4 145 464 347 483 1439 Table D.1. Clade affiliation of cmuA TRFs based on in silico analysis of database cmuA sequences.