Transcript
Page 1: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

The Biodiversity of Hydrogenases in Frankia

Characterization, regulation and phylogeny

Melakeselam Leul Zerihun

DOCTORAL DISSERTATION

To be defended on Friday 7th December 2007, 10:00 AM at

the Lecture Hall KB3A9, KBC, Umeå University

Faculty opponent

Kornel Kovacs, Professor, University of Szeged, Hungary

Department of Plant Physiology

Umeå Plant Science Center

Umeå University, Sweden

Page 2: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

©Melakeselam Leul Zerihun, 2007

Department of Plant Physiology

Umeå Plant Science Center

Umeå University

SE-901 87 Umeå

Sweden

Doctoral Dissertation, Umeå 2007

ISBN 978-91-7264-444-1

Printed by VMC, KBC, Umeå University, Umeå.

Page 3: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

“Life is available to anyone no matter what age. All you have to do is grab it”- Art Carney

Dedicated to

my beloved wife Genet F. Shawl

my beloved daughter Abigail

my beloved parents

Page 4: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

The Biodiversity of Hydrogenases in Frankia: Characterization, regulation and

phylogeny

Melakeselam Leul Zerihun (2007) ISBN 978-91-7264-444-1

Department of Plant Physiology, Umeå Plant Science Center, Umeå University, Sweden

Dissertation abstract

All the eighteen Frankia strains isolated from ten different actinorhizal host plants

showed uptake hydrogenase activity. The activity of this enzyme is further increased by

addition of nickel. Nickel also enhanced the degree of hydrogenase transfer into the

membranes of Frankia, indicating the role of this metal in the processing of this enzyme.

The uptake hydrogenase of Frankia is most probably a Ni-Fe hydrogenase.

Genome characterization revealed the presence of two hydrogenase genes

(syntons) in Frankia, which are distinctively separated in all the three available Frankia

genomes. Both hydrogenase syntons are also commonly found in other Frankia strains.

The structural, regulatory and accessory genes of both hydrogenase synton #1 and #2 are

arranged closely together, but in a clearly contrasting organization. Hydrogenase synton

#1 and #2 of Frankia are phylogenetically divergent and that hydrogenase synton #1 is

probably ancestral among the actinobacteria. Hydrogenase synton #1 (or synton #2) of

Frankia sp. CcI3 and F. alni ACN14a are similar in gene arrangement, content and

orientation, while the syntons are both reduced and rearranged in Frankia sp. EANpec.

The hydrogenases of Frankia sp. CcI3 and F. alni ACN14a are phylogenetically grouped

together but never with the Frankia sp. EAN1pec, which is more closely related to the

non-Frankia bacteria than Frankia itself. The tree topology is indicative of a probable

gene transfer to or from Frankia that occurred before the emergence of Frankia. All of

the available evidence points to hydrogenase gene duplication having occurred long

before development of the three Frankia lineages. The uptake hydrogenase synton #1 of

Frankia is more expressed under free-living conditions whereas hydrogenases synton #2

is mainly involved in symbiotic interactions. The uptake hydrogenase of Frankia can also

be manipulated to play a larger role in increasing the efficiency of nitrogen fixation in the

Page 5: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

root nodules of the host plants, there by minimizing the need for environmentally

unfriendly and costly fertilizers.

The hydrogen-evolving hydrogenase activity was recorded in only four Frankia

strains: F. alni UGL011101, UGL140102, Frankia sp. CcI3 and R43. After addition of

15mM Nicl2, activity was also detected in F. alni UGL011103, Frankia sp. UGL020602,

UGL020603 and 013105. Nickel also increased the activity of hydrogen-evolving

hydrogenases in Frankia, indicating that Frankia may have different types of hydrogen-

evolving hydrogenases, or that the hydrogen-evolving hydrogenases may at least be

regulated differently in different Frankia strains. The fact that Frankia can produce

hydrogen is reported only recently. The knowledge of the molecular biology of Frankia

hydrogenase is, therefore, of a paramount importance to optimize the system in favor of

hydrogen production. Frankia is an attractive candidate in search for an organism

efficient in biological hydrogen production since it can produce a considerable amount of

hydrogen.

Key words: Biodiversity, Frankia, immunoblotting, gene expression, uptake

hydrogenase, hydrogen-evolving hydrogenase, nickel, phylogeny

Page 6: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

CONTENTS

PAGE

LIST OF PAPERS 9

ABBREVIATIONS 10

PREFACE 11

INTRODUCTION 12

Hydrogen 12

Biodiversity of hydrogenases 13

Physiological Regulation of Hydrogenases 16

Biotechnology of hydrogenases 17

Hydrogen metabolism in nitrogen-fixing organisms 18

Nitrogenases 18

Uptake hydrogenases 18

Bidirectional/reversible hydrogenases 20

Frankia and their host plants 21

SUMMARY OF MATERIALS AND METHODS 24

Frankia strains and growth conditions 24

Seeds and inoculation of host plants 25

Enzyme activity assays 25

Protein extraction, determination and electrophoretic analysis 26

Immunoblotting and immunolabeling 27

Phylogenetic analysis of Frankia hydrogenases 28

Transcriptional analysis of Frankia hydrogenases 29

THE AIM OF THIS THESIS 30

7

Page 7: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

RESULTS AND DISCUSSION 30

UPTAKE HYDROGENASES IN FRANKIA 30

The molecular characterization of uptake hydrogenases in Frankia 31

The structure of uptake hydrogenases in Frankia 33

The phylogeny of uptake hydrogenases in Frankia 35

The regulation of uptake hydrogenases in Frankia 37

Hydrogenase gene expression in free-living vs. symbiotic condition 37

Ni-dependent regulation of uptake hydrogenases in Frankia 38

Effects of nitrogenase and hydrogen on the uptake hydrogenase of Frankia 39

HYDROGEN-EVOLVING ENZYMES IN FRANKIA 39

Molecular characterization of hydrogen-evolving enzymes in Frankia 40

Gel and peptide analysis 40

Localization of the hydrogen evolving enzyme in Frankia 41

Regulation of the enzymes in Frankia 41

Nitrogenase and the hydrogen-evolving hydrogenases of Frankia 41

Ni-dependent regulation of the hydrogen-evolving hydrogenases of Frankia 42

DOES FRANKIA HAVE OTHER HYDROGENASES? 43

CONCLUSIONS 43

FUTURE PERSPECTIVES 45

Frankia – bakterien som pruducerar både kväve gödsel och vätgas! 46

ACKNOWLEDGEMENTS 47

REFERENCE LIST 49

8

Page 8: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

List of papers The thesis is based on the publications listed below, which will be referred to in the text

by their corresponding Roman numerals.

I. Leul M, Mohapatra A and Sellstedt A (2005) Biodiversity of hydrogenases in

Frankia. Curr Microbiol 50(1): 17-23.

II. Leul M, Mattsson U and Sellstedt A (2005) Molecular characterization of uptake

hydrogenase in Frankia. Biochem Soc Trans 33: 64–66.

III. Leul M, Normand P and Sellstedt A (2007) The organization, regulation and

phylogeny of uptake hydrogenase genes in Frankia. Physiologia Plantarum

130(3): 464-70.

IV. Mohapatra A, Leul M, Mattsson U and Sellstedt A (2004) A hydrogen-evolving

enzyme is present in Frankia sp. R43. FEMS Microbiol Lett 236(2): 235-40.

V. Mohapatra A, Leul M, Sandström G and Sellstedt A (2006) Occurrence and

characterization of the hydrogen-evolving enzyme in Frankia sp. Int J Hydrogen

Energy 31: 1445-51.

VI. Leul M and Sellstedt A (2007) The phylogeny of uptake hydrogenases in

Frankia. Manuscript.

Papers I-V are reproduced with the kind permission of the publishers.

9

Page 9: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Abbreviations

Ct control LGT lateral gene transfer

PCR polymerase chain reaction

REST relative expression software tool

RT-PCR reverse transcription–polymerase chain reaction

REST relative expression software tool

ARA acetylene reduction activity

GC gas chromatograph

MALDI-TOF matrix-assisted laser-desorption-ionization

-time-of-flight mass spectrometry

NAD nicotinamide adenine dinucleotide

NADH nicotinamide adenine dinucleotide hydride

GTPase guanosine triphosphatase.

hup hydrogen uptake

shc squalene hopane cyclase

nif genes encoding nitrogenase

10

Page 10: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Preface

The increased awareness of global environmental crises and the depletion of fossil fuels

have prompted researchers to seek alternative, renewable energy sources. One of the

obvious options is hydrogen, which could potentially be used as an extremely clean

energy source, producing only water on burning. Hydrogen can be produced biologically

by microorganisms, thanks to the special group of their enzymes called hydrogenases.

Hydrogenases also increase the efficiency of nitrogen the fixation process and have other

other biotechnological applications such as wastewater treatment etc.

Hydrogenases have been characterized in detail in some organisms. In Frankia,

the research work progressed specially over the last decade as a new ways of growing

Frankia was being adopted. The recent availability of the three Frankia genomes did

definitely contributed to this study. The knowledge of the molecular biology of Frankia

hydrogenase is of a paramount importance to optimize the system in favor of hydrogen

production or nitrogen fixation. In this thesis the characterization, regulation and

phylogeny of Frankia hydrogenases have been studied, which I hope, will increase

knowledge of hydrogenases.

Melakeselam Leul Zerihun

Department of Plant Physiology

December 2007, Umeå

11

Page 11: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Introduction

There are more than 25 published definitions of biodiversity, which is short for

biological diversity. The simplest is "variation of life at all levels of biological

organization". Since the variety of life can be expressed in various ways, there is no

overall measure of biodiversity; rather there are multiple measures of different facets of

it (Gaston and Spicer, 2004). Biodiversity has traditionally been identified at three

levels: (i) genetic diversity (diversity of genes within a species); (ii) species diversity

(diversity among species in an ecosystem); and (iii) ecosystem diversity (diversity at a

higher level of organization, the ecosystem). Thus, biodiversity for geneticists is the

diversity of genes and organisms, but for ecologists it applies to the diversity of species

in the context of their immediate environments and ecosystems.

In the project this thesis is based upon the diversity of hydrogenases within the

genus Frankia was examined, rather than the diversity of the organisms per se. In

addition, Frankia hydrogenases were compared with those of other organisms, and the

regulation of Frankia hydrogenases under various environmental conditions was

investigated. Various Frankia strains were used that were originally isolated by

screening a wide range of actinorhizal host plants from diverse parts of the world. Hydrogen

Hydrogen, the simplest naturally occurring atom, is the most abundant of all the

elements, accounting for three-fourths of the mass of the universe. The abundance of

gaseous hydrogen at the earth’s surface is generally low, because it is less dense than air.

However, hydrogen is a major component of myriads of compounds, and it is found in

all organisms (biomass) in both a huge range of molecules and in the ionic form, H+ (or,

more precisely, protonated water complexes). Hydrogen also accounts for ca. 70% of the

sun’s current mass (helium accounting for a further ca. 28% and all other constituents for

<2%). The fusion of the nuclei of hydrogen atoms into helium atoms releases radiant

energy that sustains life on earth, and some of this energy is eventually stored as

12

Page 12: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

chemical energy in fossil fuels. Most of the energy we use today originates from the

sun's radiant energy. However, hydrogen is a promising energy carrier that has potential

use as an extremely clean energy source, producing only water on burning.

Hydrogen is involved in fundamental aspects of microbial physiology and it plays

a central role in life forms that inhabit anaerobic environments. It has been estimated that

about 200 million tones of hydrogen are produced and consumed per year in anoxic

habitats (Thauer et al., 1996). In spite of the high turnover rates, the steady-state

concentration of H2 in most anoxic habitats is very low (1-10 Pa), indicating that H2

formation rather than H2 consumption is the rate-limiting step in the overall process.

Hydrogen-consuming anaerobes obtain energy by using the electrons from hydrogen to

produce methane (methanogens) or acetate from carbon dioxide (acetogens), sulfide

from sulfate (sulfate reducers), ferrous from ferric iron (iron reducers), or nitrogen and

nitrite from nitrate (denitrifying bacteria) depending on the environment (Adams and

Stiefel, 1998). These and other organisms can metabolize hydrogen since they produce a

special group of enzymes called hydrogenases.

Biodiversity of hydrogenases

Hydrogenases are microbial enzymes that catalyze the reversible oxidation of molecular

hydrogen. Hydrogenase activity has been reported in a large number of anaerobic and

aerobic prokaryotes, as well as some eukaryotes, including various algae, green plants

(such as barley), and protozoa (Adams et al., 1981; Lindmark and Muller, 1973; Torres

et al., 1986).

Hydrogenases differ in the type of electron carriers they use, and associated

differences in their structures and, more importantly, redox potentials (Mertens and

Liese, 2004). Some hydrogenases reduce electron acceptors like quinones, while others

that are hydrogen producers have electron donors such as ferredoxins and cytochromes

(Cammack, 2001). Analysis of the physiological diversity of hydrogenases has revealed

three phylogenetically distinct classes: the [Fe] hydrogenases, the [NiFe] hydrogenases

and the metal-free hydrogenases (Vignais et al., 2001). The [NiFe] hydrogenases include

a subgroup of hydrogenases containing selenium called [NiFeSe]-hydrogenases (Vignais

13

Page 13: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

et al., 2001) that are probably ancient, and are only found in Archaea (Robson, 2001).

Each group is characterized by a distinctive functional core that is conserved within each

class. The widely spread and most thoroughly studied [NiFe] hydrogenases are less

active than their Fe-only counterparts, and their physiological role is usually the

oxidation of H2. The [Fe] hydrogenases are found in few microorganisms and are

difficult to study due to their sensitivity to oxygen. Their usual function is H2 evolution, and they have higher specific activities than the [NiFe] hydrogenases (Adams, 1990). It

has been noted that the term “[FeS] cluster-free hydrogenases” is more appropriate for

the third group of hydrogenases “metal-free hydrogenases”, since this group contains

functional iron, although it is not catalytically active (Lyon et al., 2004). The proteins of

the so-called metal-free hydrogenases are encoded by hmd genes and they may play

important roles in methanogenesis in nickel-deficient conditions, since their specific

activities increase in cells growing in nickel-limiting conditions (Afting et al., 1998).

Hydrogenases vary substantially in terms of subunit composition, metal content,

structure and size between different organisms (Adams, 1990). For example, the [Fe]

hydrogenase of the anaerobic bacterium Megasphaera elsdenii has only a single

polypeptide chain of 58 kDa while that of Desulfovibrio desulfuricans ATCC 7757 has

two different subunits of 42.5 kDa and 11 kDa (Filipiak et al. 1989). Hydrogenases may

also vary in the size of their structural subunits. The hydrogenase of Thermotoga

maritima contains iron as the only metal and consists of three subunits, with masses of

73 (α), 68 (β) and 19 (γ) kDa (Verhagen et al., 1999).

Further functional analyses of hydrogenases have identified 13 families to date

(Table 1), all of which, but one, are directly or indirectly involved in energy metabolism

(Robson, 2001). The main physiological functions of hydrogenases are the oxidation of

H2 or reduction of protons. The oxidation of hydrogen is linked to energy conservation,

via coupling to energy-conserving electron transfer chain reactions, allowing energy to

be obtained either from H2 or the oxidation of substrates of lower potential. The

evolution of hydrogen (H+ reduction) is linked to the disposal of excess reducing

potential. Other hydrogenases, e.g. bidirectional NAD(P)-reactive hydrogenases of

cyanobacteria, may interact with respiratory electron transport chains and provide

electron "valves" that control the redox poise of the respiratory chain at the level of the

14

Page 14: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Hydrogenase family Occurrence Function . Fe-only hydrogenases Obligately anaerobic Fermentation/ bacteria and Eubacteria Energy conservation? NAD(P)-reactive Obligately anaerobic, Fermentation hydrogenases Archaea NiFe-hydrogenases associated Facultative and obligate Fermentation with the formate hydrogen anaerobes, Archaea lyases complex NiFe(Se) membrane-bound Aerobes, facultative anaerobes, Energy conservation periplasmic hydrogenases Proteobacteria NAD(P)-reactive Facultative and obligately Energy conservation hydrogenases anaerobic Eubacteria F420-non-reactive Methanogens Energy conservation hydrogenases F420-reactive Methanogens Energy conservation hydrogenases Non metal hydrogenases Methanogens Energy conservation NiFe (thylakoid) uptake Cyanobacteria Energy conservation? hydrogenases Bidirectional NAD (P)-reactive Cyanobacteria Energy conservation, hydrogenases Eedox poising? NiFe-sensor hydrogenases Chemolithotrophic/ Hydrogen sensing Phototrophic proteobacteria components in genetic regulation of hydrogenase expression Ech hydrogenases Methanogenesis pathway Methanogenesis pathway .

Table 1. Families of hydrogenases: their occurrences and functions (Redrawn from Robson,

2001).

quinone pool and ensure the correct functioning of the respiratory chain in the presence of

excess reducing equivalents (Vignais and Colbeau, 2004; Robson, 2001). Soluble [NiFe]

hydrogenases, HupUVs, which have been identified in organisms like Rhodobacter

capsulatus (Elsen et al., 1996) and B. japonicum (Black et al., 1994) may participate in the

regulation of gene expression by acting as hydrogen sensors. The facultative

chemolithoautotroph R. eutropha also harbors a regulatory hydrogenase, HoxBC, which

enables the organism to sense hydrogen in its environment (Kleihues et al., 2000).

15

Page 15: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Physiological Regulation of Hydrogenases

Organisms may have one or more types of hydrogenases. For instance, five sets of

structural genes that code for active hydrogenases have been identified in Thiocapsa

roseopersicina (Kovács and Rákhely, 2007). The presence of multiple isofunctional

hydrogenases in some microorganisms indicates the importance of hydrogen in their

metabolism and their ability to modify their metabolism in adaptive responses to

different environments. Different hydrogenase isoenzymes may be localized in different

cell compartments: the cytosol, cell membrane or periplasm. Hydrogenases are also

known to be differentially expressed under different environmental conditions and to

have differing functions (Robson, 2001).

The ability of microbes to either take up or evolve H2 is usually a facultative trait.

A variety of factors, including the concentration of hydrogen, oxygen, nickel ions,

molybdenum, nitrate, formate, carbon monoxide, nitrogen/phosphate, carbon and energy

sources can affect hydrogenase gene expression (Friedrich et al., 2001; Vignais and

Colbeau, 2004). Molecular hydrogen, which is also the substrate, activates hydrogenase

expression in aerobic bacteria, photosynthetic bacteria and free-living Rhizobia, whereas

molecular oxygen is inhibitory for most hydrogenases. Hydrogenase synthesis in

facultatively H2-oxidizing bacteria like Azotobacter vinelandii (Kennedy and

Toukdarian, 1987) and Bradyrhizobium japonicum (Hanus et al., 1979) depends on the

availability of H2. In facultative and (especially) obligately anaerobic bacteria, the

availability of O2 and the redox state of the cells are important regulatory variables for

hydrogenase gene expression (Kovács et al., 2005). Several carbon sources, such as

pyruvate and propionate, have also been shown to affect the regulation of hydrogenase

activity in three strains isolated from Casuarina sp. (Sellstedt et al., 1994). The

heterotrophic and strictly anaerobic archaeon Methanococcus voltae harbors four

hydrogenase operons, including two encoding [NiFe] hydrogenases that are expressed

under selenium depletion conditions when [NiFeSe] hydrogenases cannot be made in

sufficient amounts (Berghofer et al., 1994). Hydrogenase gene expression in A.

cylindrica sp. strain PCC7120 requires genome re-arrangements that occur during the

cellular differentiation process leading to heterocyst formation (Friedrich et al., 2001).

16

Page 16: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Biotechnology of hydrogenases

Knowledge about hydrogenases in microorganisms has greatly increased in the last

decade, and our enhanced understanding of the structure and function of the active sites

of hydrogenases has led to the synthesis of a close analogue of the hydrogen-producing

active centre of hydrogenases, the H-cluster (Tard et al., 2005). The availability of an

active, free-standing analogue of the H-cluster has enabled scientists to develop useful

electrocatalytic materials for applications in, inter alia, reversible hydrogen fuel cells.

The precious metal platinum (Pt) is the currently preferred electrocatalyst for such

applications, but it is very expensive (costing more than $17 per gram), its availability is

limited and its use is unsustainable in the long term. Thus, such alternatives could be

extremely valuable. Hydrogenases can also be used in various biotechnological

applications such as biohydrogen production, wastewater treatment, the prevention of

microbial-induced corrosion and the generation/regeneration of NADP cofactors

(Mertens and Liese, 2004).

In addition, increasing awareness of global environmental crises and the depletion

of fossil fuels has prompted researchers to seek alternative, renewable energy sources.

An obvious option is hydrogen, which could potentially be used as an extremely clean

energy source, producing only water on burning. Biological hydrogen production by

photosynthetic prokaryotic and eukaryotic organisms (e.g. cyanobacteria and the green

alga Chlamydomonas reinhardtii) or by fermentation in anaerobic bacteria (e.g.

Clostridium butyricum) has been reported (Melis et al., 2000; Karube et al., 1976).

Several strategies for boosting their hydrogen production are being explored, such as

genetic modification of the light-harvesting antennae complexes, sulfur deprivation of

the cultures, screening for mutants that produce more hydrogen than wild type strains,

optimization of conditions for the hydrogenase enzyme and investigation of naturally

occurring hydrogen production. Biological hydrogen production has several advantages

over conventional means of hydrogen production, such as photoelectrochemical or

thermochemical processes.

17

Page 17: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Hydrogen metabolism in nitrogen-fixing organisms

Three key classes of enzymes that are directly involved in hydrogen metabolism have

been identified in nitrogen-fixing organisms to date: nitrogenases, uptake hydrogenases

and bidirectional hydrogenases (Fig. 1). More than 10 million tons of H2 are globally

generated in oxic habitats (e.g. oxic soils and fresh water) by aerobic and

microaerophilic microorganisms as side-products of nitrogen fixation (Thauer et al.,

1996).

Nitrogenases: are oxygen-labile enzymes that catalyze the reduction of nitrogen to

ammonia in the highly energy-demanding process of nitrogen fixation, which requires

metabolic energy in the form of ATP as shown in the reaction:

N2 + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi

Since two ATP molecules are required for each electron transferred from dinitrogenase

reductase to dinitrogenase, a total of 16 ATP molecules are needed to reduce dinitrogen

(N2) to ammonia (NH3), a form in which the nitrogen is available for further biological

reactions. During the nitrogen fixation process, substantial amounts of hydrogen are

produced via the reduction of protons, catalyzed by the nitrogenase enzyme. In fact, it

has been shown that in most symbionts only 40-60% of the electron flows to the

nitrogenase are transferred to nitrogen, and the remainder is lost through hydrogen

evolution (Schubert and Evans, 1976).

Uptake hydrogenases: Uptake hydrogenases catalyze the consumption of hydrogen (H2

oxidation) produced by nitrogenases during nitrogen fixation. Hydrogen oxidation is

coupled to the reduction of electron acceptors such as oxygen, nitrate, sulfate, carbon

dioxide, and fumarate. The hup (hydrogen uptake) systems have been studied in detail in

two species of root nodule rhizobia, Rhizobium leguminosarum bv. viciae and B.

japonicum, from which a multigenic (18–24 genes) cluster responsible for the synthesis

18

Page 18: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

of an active hydrogenase has been isolated (Ruiz- Argüeso et al., 2000). Uptake

hydrogenase is considered beneficial to the nitrogen-fixing organisms in both the free-

living and symbiotic states (Dixon, 1976) since the hydrogen produced during the

nitrogen fixation can be consumed and the reductant generated can be used by the cells

in various ways. Hydrogen recycling has been shown to reduce energy losses associated

with nitrogen fixation (Schubert and Evans, 1976). In addition to the provision of an

additional source of energy, other possible functions such as prevention of H2 inhibition

of the nitrogenase reaction and protection of oxygen-sensitive nitrogenase from O2

damage have been proposed (Dixon, 1972). All strains of cyanobacteria (Tamagnini et

al., 2002) and Frankia (Sellstedt, 1989) investigated to date have uptake hydrogenases,

but only a few examined strains of rhizobia have this enzyme.

Fig. 1. Enzymes directly involved in hydrogen metabolism in cyanobacteria (Redrawn

from Tamagnini et al., 2002). The bidirectional hydrogenase of cyanobacteria has five

subunits: HoxEFUYH (Schmitz et al., 2002).

Uptake hydrogenase and nitrogenase encoding genes of Rhi. leguminosarum,

which are induced together, are controlled by the nitrogen fixation regulatory protein

19

Page 19: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

NifA (Brito et al., 1997). A strong correlation between activities of nitrogenase and

uptake hydrogenase has been reported in Frankia, although they might not be

coregulated (Mattsson and Sellstedt, 2000). Also, uptake hydrogenase was localized in

vesicles and hyphae (Sellstedt and Lindblad, 1990).

Bidirectional/reversible hydrogenases: this group of hydrogenases has the capacity to

metabolize hydrogen both directions. They have been characterized by their sensitivity

to oxygen, thermotolerance, and high affinity to hydrogen, and are widely distributed

among cyanobacteria, including nitrogen fixing, non-nitrogen-fixing, unicellular, non-

heterocystous, and heterocystous strains (Houchins, 1984). The physiological functions

of the bidirectional hydrogenases are still unclear but it has been suggested that they may

mediate the release of excess reducing power in anaerobic environments (Tamagnini et

al., 2002), acting as electron valves during the light reactions of photosynthesis and thus

preventing retardation of the electron transport chain under stress conditions (Appel et

al., 2000), and/or be involved in fermentation (Troshina et al., 2002) and respiratory

complex I (Appel et al., 1996). Since the activity of the enzymes is not strongly affected

by combined hydrogen, it has also been suggested that these hydrogenases may function

independently of nitrogen fixation (Tamagnini et al., 2002). Furthermore, it has been

shown that the biosynthesis of nitrogenase is not essential for biosynthesis of the

bidirectional hydrogenase and hydrogen evolution in several unicellular strains (Howarth

and Codd, 1985).

The crystal structures of the [Ni-Fe] hydrogenases of five sulfate-reducing bacteria

(Desulfovibrio gigas, D. vulgaris, D. desulfuricans, D. fructosovorans and

Desulfomicrobium baculatum) have been reported so far (Volbeda et al., 1995; Higuchi

et al., 1997; Matias et al., 2001; Montet et al., 1997; Garcin et al., 1999). The crystal

structures of the [FeFe] hydrogenases of two organisms (Clostridium pasteurianum and

D. desulfuricans) have also been resolved (Peters et al., 1998; Nicolet et al. 1999). Both

NiFe and Fe-hydrogenases share a common active site low-spin Fe center with CO and

CN coordination, although these hydrogenases are evolutionarily unrelated. From the

studies undertaken to date, [Fe] hydrogenases appear to have simpler structures than the

[NiFe] hydrogenases (Nicolet et al., 2002).

20

Page 20: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Frankia and their host plants

Frankia is a genus of nitrogen-fixing filamentous, heterotrophic, Gram-positive,

actinomycetous soil bacteria. Frankia resemble fungi and are phylogenetically and

morphologically distinct from the rhizobial bacteria that are responsible for nitrogen

fixation in legumes (Binkley et al., 1994). Frankia can fix nitrogen in both free-living

aerobic conditions and in symbiosis, unlike other soil microsymbionts, such as some

species of rhizobia (Zhang et al., 1984). Frankia can differentiate into three cell types:

hyphae, vesicles and spores. The vesicles are formed under nitrogen-limiting conditions

from the swollen tips of hyphae in most free-living Frankia. Vesicles, in which the

oxygen-sensitive nitrogenase is localized (Meesters, 1987; Huss-Danell and Bergman,

1990), are surrounded by a multi-layer lipid membrane that maintains a low internal

oxygen tension (Parsons et al., 1987). In a liquid medium, an exponentially growing

culture forms spherical or ellipsoidal mycelial colonies, while overgrown cultures are

like huge, uniform mycelia (Schwencke, 2001).

The first attempt to classify members of the genus Frankia was by Baker (1987),

who proposed that there were four “infectivity groups”, based on the results of

infectivity studies using pure cultures in cross-inoculation tests, although it is

questionable whether such tests reflect host specificity under normal conditions. A more

sophisticated approach based on phenotypic characteristics was subsequently adopted,

which differentiated two Frankia species; F. alni and F. elaeagni (Lalonde et al., 1988).

In the following years, phylogenetic studies were performed, based on analyses of the

widely used 16S rDNA and 16S rRNA sequences (Nazaret et al., 1991; Normand et al.,

1996; Clawson et al., 2004), arbitrary primers (Sellstedt et al., 1992), nitrogen fixation

genes (Jeong et al., 1999) and glutamine synthetase (Clawson et al., 2004). The studies

conducted to date indicate that Frankia strains can be generally divided into three

clusters. Cluster 1 includes strains that nodulate plants of the Fagales, Betulaceae and

Myricaceae and are often referred to as “Alnus strains” (Normand et al., 1996) and a

subclade of “Casuarina strains”, which only nodulate Casuarina and Allocasuarina

species of the Casuarinaceae under natural conditions (Benson et al., 2004). Cluster 2 is

comprised of unisolated strains of Frankia that only infect members of the Coriariaceae,

21

Page 21: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Datiscaceae, Rosaceae and Ceanothus of the Rhamnaceae. Cluster 3 strains form

effective nodules on members of the Myricaceae, Rhamnaceae, Elaeagnaceae and

Gymnostoma of the Casuarinaceae. In the studies underlying this thesis the phylogenetic

relationships and characteristics of Frankia hydrogenases (and their relationships with

hydrogenases of other organisms), rather than those of Frankia organisms, were

investigated. In all cases in the following text the term “hydrogenase synton” will be

used instead of “hydrogenase cluster” to avoid possible confusion with the term

“Frankia cluster” described in this paragraph.

Frankia can interact and form symbiotic relationships with a diverse, globally

distributed group of dicotyledonous plants called actinorhizal plants that are classified

into four subclasses, eight families, and 25 genera of plants comprising more than 240

species of dicotyledonous angiosperms (Wall, 2000). Actinorhizal plants are widespread

(Fig. 2) and grow in all types of climate, although they are mainly found in temperate

climates (Silvester, 1977). They inhabit diverse ecosystems, including arctic tundra

(Dryas species), coastal dunes (Casuarina, Hippophae, Myrica, and Elaeagnus species),

riparian (Alnus and Myrica species), glacial till (Alnus and Dryas species), forest (Alnus,

Casuarina, Coriaria, and Shepherdia species), chapparal and xeric (Casuarina, Purshia,

Ceanothus, Cercocarpus, Comptonia, and Cowania species), and alpine (Alnus species)

systems (Benson and Silvester, 1993). Actinorhizal plants often serve as pioneer species

in early successional plant communities since they thrive on marginal soils. They

contribute considerable amounts of fixed nitrogen, especially in cool, temperate areas

where indigenous legumes are rare or absent (Silvester, 1976). They are economically

important in forestry programs, such as land reclamation and reforestation programs, and

have high potential for introduction in areas with problem (arid, saline or waterlogged)

soils, for timber, pulp and fuel wood production, and for acting as windbreaks and/or

ornamental plants (Chaudhary and Mirza, 1987; Diem and Dommergues, 1990).

22

Page 22: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Fig. 2. Present-day native distribution of actinorhizal plant hosts: (I) Betulaceae (B) and

Myricaceae (M) and their overlap (M+B); (II) Elaeagnaceae (E), Myricaceae (M),

Rhamnaceae (R). Elaeagnaceae and Myriceae (E+M) overlap in some areas. The

Casuarinaceae (not shown in the figure) are distributed in Indo-Malaysia, Australia, and

the Pacific islands (Redrawn from Normand et al., 2007a)

23

Page 23: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Summary of materials and methods

Frankia strains and growth conditions

Twenty Frankia strains isolated from 12 different actinorhizal host plants native to

different parts of the world (Table 2) were grown at 27ºC, as described in Mattsson and

Sellstedt (2000). Cells were successively transferred to fresh PUM medium containing

0.1 g/L NH4Cl on a weekly basis to obtain actively growing cultures. In the experiments

Frankia strains Source or references Location Host plant . F. alni ACN14a Normand and Lalonde, 1982 Canada A. viridis subsp. crispa

F. alni AvCI1 Baker and Torry, 1980 USA A. viridis subsp. crispa

Frankia sp. KB5 Sellstedt et al., 1991 Australia C. equisetifolia

Frankia sp. UGL020603 Vel´azquez et al., 1998 Egypt C. equisetifolia

Frankia sp. UGL020602 Wheeler C Brazil C. equisetifolia

F. alni UGL011103 Wheeler C Sweden A. incana

F. alni UGL011102 Wheeler C Sweden A. incana

F. alni ArI3 Berry and Torrey, 1979 USA A. rubra

Frankia sp. 013105 Wheeler C USA A. rubra

F. alni 010701 Wheeler C Scotland A. glutinosa

F. alni 010702 Wheeler C Scotland A. glutinosa

F. alni UGL011301 Sayed et al., 1997 S. Korea A. inokumai

Frankia sp. UGL161101 Wheeler C Scotland M. gale

Frankia sp. UGL161102 Wheeler C Scotland M. gale

Frankia sp. HFPCcI3 Zhang et al., 1984 USA C. cunninghamiana

Frankia sp. R43 Zhang et al., 1984 USA C. cunninghamiana

Frankia sp. EAN1pec Lalonde et al., 1981 USA E. angustifolia

Frankia sp. BCU110501 Chaia, 1998 Argentina D. Trinervis

Frankia sp. UGL140104 Wheeler C Scotland H. rhamnoides

Frankia sp. UGL140102 Lumini et al., 1996 Scotland H. rhamnoides

Frankia sp. CpI1 Callaham et al., 1978 USA C. peregrina .

Table 2. Frankia sp. strains used in the study.

24

Page 24: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

cells were placed in portions of 50 mL growth medium in 100 mL flasks, at 5 μg/mL

total protein concentration, without nitrogen to induce vesicle formation and nitrogen

fixation, to which nickel (II) chloride was added at various concentrations, where

appropriate, to assess the effects of nickel on hydrogenase expression.

Seeds and inoculation of host plants

Alnus glutinosa seeds were sterilized, imbibed in water overnight and germinated before

being transferred to a plastic pot containing a sterile soil and vermiculite mix (5:1 ratio),

which was supplemented with Evans solution (Evans et al., 1972) twice a week. When

the seedlings were six weeks old they were inoculated with 5 ml (from a 5 mg protein

per ml bacterial culture) of F. alni ACN14a. The plants were grown in a growth chamber

with metal halide lamps (HQI-T, 400 W, daylight) providing light with an irradiance of

300 μmol m-2s-2 for 17 h day-1. The day/night temperature was kept at 20ºC/17ºC and the

relative humidity at 70%.

Enzyme activity assays

Nitrogenase assays: nitrogenase activity in cultures of free-living Frankia cells was

determined as acetylene reduction activity (ARA) using a gas chromatograph (GC-8AIF,

Shimadzu Scientific Instruments Inc., Columbia, MD), as described earlier (Mattsson

and Sellstedt, 2000).

Uptake hydrogenase: Eight-day-old Frankia cultures were collected in 6.5-mL flasks

containing 1.8 mL 50 mM Tris-HCl and sealed with a gas-tight rubber membrane.

Uptake hydrogen activity was analyzed immediately after the addition of hydrogen gas

(1% v/v) into the gas phase of the flasks and then at 1 h intervals while the strains were

incubated at room temperature with shaking, using a gas chromatograph (GC8AIT,

Shimadzu Scientific Instruments, Colombia, MD) according to Mattsson and Sellstedt

(2000).

25

Page 25: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Hydrogen-evolving hydrogenase: Frankia cultures were incubated for 24 hours under

anaerobic conditions, at 27ºC with shaking in 6.5 mL glass vials sealed with gas-tight

rubber membranes. At the start of induction of hydrogen evolution by argonization,

ammonium chloride was added to the cultures to a final concentration of 10 mM to block

hydrogen evolution from nitrogenase. Hydrogen evolution was then measured in 2 mL

reaction mixtures, each containing 1.8 mL of Frankia culture resuspended in 50 mM

Tris-HCl (pH 7.0), to which 2 mM of freshly prepared methyl viologen and 20 mM

sodium dithionite were added (Tamagnini et al., 1997). Measurements began after an

incubation period of 90 min and continued until a linear increase in hydrogen was

recorded, using gas chromatography as outlined above.

NAD-reducing hydrogenase: Hydrogen evolution from NAD-reducing hydrogenase in

Frankia sp. R43 was measured by adding NAD to cultures of the organism, and

measuring NADH formation at 340 nm as previously described (Friedrich et al., 1980).

Protein extraction, determination and electrophoretic analysis

Total protein determination: To measure their protein concentrations, Frankia cells

were collected by centrifugation, treated as described by Mattsson and Sellstedt (2000),

and the protein contents of the resulting suspensions were determined using the

bicinchonic acid (BCA) assay and BSA as a standard.

One and two-dimensional gel electrophoresis: For one-dimensional analyses, Frankia

protein extracts containing 30 μg membrane (total) proteins were electrophoretically

separated on NuPage 12% Bis-Tris gels. For two-dimensional analyses Frankia proteins

were initially precipitated by acetone, centrifuged and the resulting pellets were air-

dried. Immobilized pH gradient gels (ZOOMTM Strips) were rehydrated at room

temperature, and a ZOOMTM IPGRunner System (Invitrogen) was then used for

isoelectric focusing by gradually increasing the voltage and maintaining the final

focusing voltage for approx. 2 h. The electro-focused IPG strip was incubated in a

reducing solution for 15 min, then in an alkylating solution for 15 min before

26

Page 26: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

electrophoretic separation of the proteins in it on a NuPAGETM 4–12% Bis-Tris gel at

200 V for 50 min.

Immunoblotting and immunolabeling

Western blots analysis: The polypeptides on 1D or 2D gels were electrotransferred to

nylon transfer membrane and western blots were performed using a Western Breeze kit

(Invitrogen), according to the manufacturer’s instructions, except that the membrane was

incubated for 1-1.5 h with the primary antibody. The primary antibodies used in these

experiments were raised in rabbit against the large subunit of Ni-Fe hydrogenase (HoxG)

of the MB hydrogenase and HoxH of the SH-hydrogenase HY of R. eutropha, the small

hydrogenase subunit of B. japonicum (Hup S), and [Fe]-hydrogenase from D.

desulfuricans ATCC 7757, and were used at dilutions of 1:1000.

Southern blot analyses: Frankia DNA was digested with BamHI or Sal1, transferred to

a membrane and hybridized at 52ºC with a P32-labeled PCR fragment from part of the

small hydrogenase subunit originating from Frankia local source.

Preparation and immunolabeling of cryosections: Fixation, embedding, sectioning and

immunogold labeling were all performed as described earlier (Wheeler et al., 1998;

Mattsson et al., 2001), except that in the studies underlying this thesis we used primary

antiserum raised against HoxH and HoxG of R. eutropha, followed by secondary goat-

anti-rabbit IgG conjugated with 5 nm colloidal gold particles, before viewing the

samples under a Philips CM 10 transmission electron microscope operating at 60 kV.

Cells labeled with only the secondary antibody were used as controls.

Peptide analysis: Protein spots were excised from two-dimensional gels and cleaved in-

gel by trypsin. Peptide analysis was performed by electrospray ionization mass

spectrometry (Wilm et al., 1996) using a quadrupole-time-of-flight instrument and

Masslynx software or matrix-assisted laser-desorption ionization–time-of-flight mass

27

Page 27: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

spectrometry, in which the resulting peptide ‘fingerprints’ were analyzed using Protein

Prospector software.

Phylogenetic analysis of Frankia hydrogenase

DNA extraction: Genomic DNA was extracted from seven-day-old Frankia cultures

using the bacterial protocol supplied with the Blood and Tissue Genomic DNA

Extraction Kit (Viogene, USA).

RNA isolation and cDNA synthesis: RNA was extracted using a RNeasy Mini Kit

according to the manufacturer’s protocol. F. alni cells (4.5 days old) grown in nitrogen-

fixing conditions and fresh nodules collected from a 6-month-old plant were treated

immediately after harvest with RNA ProtectTM Bacteria Reagent (Qiagen) to stabilize the

RNA in the bacterial cells. To remove the DNA, the extracted RNA was treated with

DNA-freeTM prior to cDNA synthesis using an iScriptTM cDNA Synthesis Kit according

to the manufacturer’s (Ambion) protocol.

PCR: Fragments of genes (800 bp long) encoding structural subunits of hydrogenases

from several Frankia strains were amplified by touchdown PCR using designed primers

listed in Table 3. The PCR was performed with 25 ng of DNA in 20 µl mixtures (0.6 µM

of each primer, 3 mM of either MgCl2 or Q solution, and 1 U Taq from Qiagen, with

temperature programs of 3 min at 95 oC followed by 35 cycles of 30 s at 95 oC, 15 s at 58

oC, 15 s at 54 oC and 1 min at 72 oC, then a final elongation step of 10 min at 72 oC. PCR

products were electrophoretically separated on agarose gels, purified using a QIAquick

Gel Extraction Kit (Qiagen) and sequenced by an ABI377 sequencer (Applied

Biosystems).

Sequence alignments and phylogenetic analysis: Multiple alignments of hydrogenase

sequences of several Frankia strains and other related organisms were constructed using

ClustalX (Thompson et al., 1997). Matrix pair-wise comparisons were corrected for

multiple-base substitutions according to the method of Kimura (1980), followed by a

28

Page 28: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

phylogenetic analysis using Neighbor-Joining (Saitou and Nei, 1987) with standard

parameters. Bootstrap confidence analysis was performed using 1000 replicates to

determine the reliability of the distance tree topologies obtained (Felsenstein, 1985).

Tree representations were constructed by Tree-View (Page, 1996).

Product Designation Forward (5’- 3’) Reverse (5’ to 3’) .

HupL1 HupL 20/HupL 21 cctcgttgacccagtccttg cgcatcatcggcaacctc

HupL1 HupL 20/HupL-13B cctcgttgacccagtccttg aaggggaaggatccacgcgacgcc

HupS1 HupS-6F/HupS-6B gttgtgccgccacctcggctc tgcgacggcgacacggtctcg

HupS1 HupS-6F/HupL 24 gttgtgccgccacctcggctc acggtccacctgcacaacaa

HupL2 HupL-F1/HupL33-2-(b) gacgtcacccactcgttctac cgttgatgacgaacctgct

HupL2 HupL32-2-(f)/HupL33-2-(b) tcacccactcgttctacgc cgttgatgacgaacctgct

HupS2 HupS34-2-(f)/HupS-B2 gatgtcatccgtgctctgg agccgaactcgtagaacagg

HupS2 HupS-F1/HupS35-2-(b) tcatccgtgctctggtttc gtgggtgaacgtggtgaag

HupS2 HupS34-2-(f)/HupSB1 gatgtcatccgtgctctgg gtcggtgatcaggtcgatg

HupL2 HupL-F1/HupL(II)_B3 gacgtcacccactcgttctac gacttggcccagctgtactt

HupL1 HupS(I)-27F/HupS(I)-27B acaccaggttgtcctggaag gtgttcatgaaggggaagga

HupS1 HupS(I)_26_F3/HupL(I)-26B caccgttgatgttctcgttg ctgcacaacaaggtgctctc

16S rDNA 16S-F134/16S-B134 gatttatcggctcgggatg gtaggagtctgggccgtgt .

Table 3. List of primers used in the study.

Transcriptional analysis of Frankia hydrogenases

Real-time PCR and conventional RT-PCR: Gene transcripts were measured by

amplification using primers specific for the structural subunits of F. alni strain ACN14a

hydrogenase. The primers (Table 3) were designed using Primer Premier 5 software.

Triplicate amplification of all standards, unknowns and controls was performed using a

multicolour iCycler iQ Real-Time PCR Detection System. Unknowns were compared

with cDNA standards covering four dynamic ranges obtained by serial dilution of

quantified starting concentrations. Expression levels were calculated from standard

curves generated during each run and with each primer pair. The acquired real-time PCR

data were analyzed by the relative expression software tool REST© (Pfaffi et al., 2002).

29

Page 29: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Conventional PCR was performed in varying conditions, as optimized for the sequences

and primers concerned, and in some cases by lowering the annealing temperature

gradually from 59ºC to 56ºC.

The aim of this thesis

• To study the diversity of hydrogenases in Frankia isolated from different

actinorhizal species growing in different regions.

• To characterize the hydrogenases of Frankia at the molecular level (gene/peptide

sequencing etc).

• To study the evolutionary relationships between uptake hydrogenases of Frankia

isolated from a various different actinorhizal plants from growing in different

regions, and also in comparison with hydrogenases of other organisms.

• To study the regulation of Frankia hydrogenases under different physiological

conditions.

Results and discussion

Uptake hydrogenases in Frankia

The biodiversity project this thesis is based upon was started by screening 18 Frankia

strains originally isolated from ten different actinorhizal host plants for physiological

activity of uptake hydrogenases at day eight of their growth in a medium with no

nitrogen (nitrogen-fixing conditions), in which the activity was expected to be maximal

(Mattsson and Sellstedt, 2000). The physiological activity data were subsequently

confirmed by molecular biology techniques, including blotting (Western, Southern),

30

Page 30: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

mass spectrometry and PCR as discussed in the following sections. Hydrogenase activity

was detected in all strains investigated (Paper I), corroborating earlier studies in which

activity was found in Frankia capable of infecting Alnus in northern Sweden (Sellstedt,

1989) and Casuarina (Sellstedt et al., 1991).

The molecular characterization of uptake hydrogenases in Frankia

Western analysis: To confirm the physiological findings, several Frankia strains were

screened to investigate whether their uptake hydrogenases are immunologically related

to hydrogenases from other organisms. Antibodies rose against the large subunit of Ni-

Fe hydrogenase of R. eutropha (HoxG) recognized a polypeptide at about 60 kDa,

corresponding to the large hydrogenase subunit, in Frankia UGL020603, KB5 and

AvCI1. The HoxG antibody also recognized hydrogenases of other strains such as

Frankia sp. UGL140102 (Paper I). These results do not prove the absence of uptake

hydrogenases in other Frankia strains, but rather indicate the diversity of uptake

hydrogenases in Frankia. For example, antibodies raised against the large subunit of the

[NiFe] hydrogenase of R. eutropha recognized the large subunit of hydrogenase of

Frankia sp. KB5, but antibodies raised against the large subunit of [NiFe] hydrogenase

of A. vinelandii did not (Mattsson et al., 2001), and no hydrogenase subunits in any of

the Frankia strains investigated to date appear to be immunologically related to the small

subunit of the Ni-Fe hydrogenase (Hup S) in B. japonicum (Paper I). In the study of the

interspecies immunological cross-reactivity of hydrogenases, in seven cases the

immunological tests showed between-strain cross-reactivity with the large hydrogenase

subunits but not with the small subunits, suggesting that at least one conserved protein

region is present among the large subunits of these enzymes, while their small subunits

are less conserved (Kovács et al., 1989).

Peptide analysis: Protein spots of Frankia sp. KB5, corresponding to 60 kDa of the large

subunit of uptake hydrogenase, were excised from Coomassie-stained 2-D mini gels and

analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry

(MALDI-TOF). The resulting peptide ‘fingerprint’ showed identity (20% matches) with

31

Page 31: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

the membrane-bound hydrogenase 2 large subunit (HYD2) in Escherichia coli (Acc.

P37181). The matched peptides covered 83% (476/567 amino acids) of the protein

(Paper II).

PCR and southern blot analysis: An NCBI blastx search with the translated sequence of

a 127-bp PCR-amplified gene fragment from F. alni AvCI1yielded up to 76% similarity

with the large hydrogenase subunit of various other organisms, e.g. Azotobacter

chrococcum (Paper II).

Uptake hydrogenase activity has been recorded from all Frankia strains

investigated but one, namely Frankia “local source” (Sellstedt et al., 1986; Paper I).

However, interestingly, a partial sequence of 500 bp could be amplified from DNA

isolated from nodules of the symbiosis between Frankia ‘local source’ and A. incana

(Mattsson, 2001; Paper VI), which was analyzed using the NCBI-translated query versus

the protein database (blastx), yielding 69% and 67% identity with the small subunits of

hydrogenases of B. japonicum and Rhi. leguminosarum, respectively. In addition, using

Southern-blot analysis, the hupS fragment of Frankia ‘local source’ hybridized with

DNA isolated from Frankia sp. KB5 (Paper VI). However, the Frankia DNA used was

extracted from nodules in which several different Frankia strains may be present. It is

possible that the uptake hydrogenase of Frankia “local source” may be active only under

specific environmental conditions, which are not yet known, or that it has an uptake

hydrogenase system but some regulatory genes are missing that are required to make an

active enzyme. It has been reported that Rhi. leguminosarum bv. viciae is unable to

express its uptake hydrogenase in free-living conditions because it contains a defective

hoxA gene (Brito et al., 1997). A hup-specific transcriptional activator encoded by the

hoxA gene is known to control hydrogenase gene expression in A. eutrophus (Friedrich

and Schwartz, 1993) and B. japonicum (Van Soom et al., 1993).

The recent release of the sequences of three Frankia genomes: F. alni ACN14a

(GenBank accession no. CT573213), Frankia sp. HFPCCi3 (GenBank accession no.

CP000249) and Frankia sp. EAN1pec (GenBank accession no. ZP_00571168) by

Genoscope (France), the National Science Foundation (USA) and the U.S. Department

of Energy Joint Genome Institute, respectively, have provided a wealth of information

32

Page 32: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

and opportunities for studying Frankia hydrogenases (Normand et al., 2007a). Although

some standard genetic techniques cannot be applied to Frankia as yet (since Frankia has

never been genetically transformed), rapidly developing molecular biology techniques,

such as gene and protein arrays, could be exploited to make use of the available data and

study the molecular biology of Frankia. Further characterization of Frankia

hydrogenases (following the availability of the Frankia genome sequences) will be

addressed in the following sections.

The structure of uptake hydrogenase genes in Frankia

The genome analyses have shown the presence of two hydrogenase syntons in Frankia,

which are distinctly separated in all three genomes (Paper III). The structural, regulatory

and accessory genes of the hydrogenases are arranged closely together in each synton. In

F. alni ACN14a, hydrogenase synton #1 corresponds to GI:111221817–111221829 and

is situated at co-ordinates 2614407–2627969, whereas hydrogenase synton #2,

corresponds to GI:111221263–11221273 and is situated at co-ordinates 1959070–

1971271. Hydrogenase syntons #1 and #2 of Frankia sp. CcI3 and Frankia sp. EAN1pec

correspond to GI:86740641–86740652 and GI:86739780–86739790 and GI:68199305–

68199315 and GI:68232162–68232167, respectively (Fig. 3; Paper III). The gene

encoding the small subunit of the hydrogenase is located upstream of the large subunit in

Frankia, in accordance with several other organisms like Nostoc sp., Rhi.

leguminosarum and B. japonicum (Przybyla et al., 1992; Vignais and Toussaint, 1994;

Voordouw, 1992; Wu and Mandrand, 1993).

All of the available evidence indicates that hydrogenase syntons #1 and #2 are

both uptake hydrogenases that have many features in common with uptake hydrogenases

of other Frankia strains or other organisms. For example, the gene structures of

hydrogenase syntons #1 and #2 of F. alni ACN14a are very similar to those of

hydrogenase syntons #1 and #2 of Frankia sp. CcI3, respectively. The uptake

hydrogenases of Frankia may also have some dissimilarity, even in the same Frankia

strain. For example, the physical position and orientation of the uptake hydrogenase

genes varies very clearly between hydrogenase syntons #1 and #2 in F. alni ACN14a,

33

Page 33: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Frankia sp. CcI3 and EAN1pec. Interestingly, other symbiotic gene clusters that have

been found in the Frankia genome: the nitrogen fixation (nif) and squalene hopane

cyclase (shc), have similar numbers of genes in similar arrangement in F. alni ANC14a,

Frankia sp. CcI3, and EAN1pec (Normand et al., 2007b). In addition, a simple sequence

comparison indicated that the sequence conservation between the structural subunits of

hydrogenase syntons #1 and #2 in F. alni ACN14a itself (e.g. HupL1 of synton #1 vs.

HupL2 of synton #2) was as low as 27%, whereas that between F. alni ACN14a and

non-Frankia bacteria such as S. avermitilis was as high as 73%. Clearly, one of the

syntons is not simply a duplicate of the other, but rather both are required for hydrogen

metabolism under different circumstances in Frankia (Paper III).

Fig. 3. Genome maps of Frankia sp. EAN1pec and CcI3. Circles, from the outside in,

show (1) gene regions related to symbiosis including shc1, hup2, hup1, and nif; (2) the

coordinates in Mb beginning at 0 = oriC; (3) regions of synteny (syntons) calculated as a

minimum of five contiguous genes present in all strains with an identity >30% over 80%

of the length of the shortest gene. Redrawn from Normand et al., 2007a.

34

Page 34: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

The phylogeny of uptake hydrogenases in Frankia

The phylogenetic analysis of the structural subunits hydrogenase syntons #1 and #2 of F.

alni ACN14a, Frankia sp. CcI3 and EAN1pec has shown that the two syntons are

distinctly different. This is not surprising considering the degree of sequence divergence

between them, even in the same Frankia strain. The phylogenetic and sequence

similarity of one of the hydrogenase syntons to hydrogenases of other organisms, on the

other hand, is remarkable.

According to the phylogenetic trees of uptake hydrogenase syntons #1 and #2, the

structural subunits of F. alni ACN14a and Frankia sp. CcI3, which belong to

phylogenetic Frankia cluster 1, group together but not with those of Frankia sp.

EAN1pec, which belongs to Frankia cluster 3 (Normand et al., 1996). HupL2 of

Frankia sp. EAN1pec appears to be most closely related to the hydrogenases of the non-

Frankia bacteria Geobacter Sulfurreducens, providing strong evidence for the

occurrence of lateral gene transfer (LGT) between these organisms. Clearly, neither of

the syntons is simply a recent duplicate of the other, and functional complementarities

are less likely, given their apparent sequence divergence. The tree topology is indicative

of probable gene transfer to or from ancestral organisms that occurred before the

emergence of Frankia. All of the available evidence points to hydrogenase gene

duplication having occurred long before emergence of the three Frankia lineages (Paper

III).

Phylogenetic analysis of the structural subunits of hydrogenase syntons #1 and #2

(analyzed separately) showed distinct clustering among hydrogenases of various Frankia

strains (Paper VI). The large subunits of hydrogenase synton #1 of Frankia sp. CpI1, F.

alni ACN14a and AvCI1 (isolated from A. viridis subsp. crispa) grouped together, while

those of Frankia sp. CcI3, KB5, UGL140104 and UGL011102 (isolated from C.

cunninghamiana, C. equisetifolia, H. rhamnoides and A.incana, respectively) formed

another group (Table 4). The large subunits of hydrogenase synton #2 of F. alni

ACN14a, Frankia sp. CcI3 and BCU110501 (isolated from D. trinervis) also grouped

together, while those of Frankia sp. KB5, CpI1, F. alni AvCI1 and ArI3 (isolated from

35

Page 35: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Hydrogenase synton Phylogenetic groups Frankia strains . # 1 A Frankia sp. CpI1, Frankia sp. AvCI1,

F. alni ACN14a

B Frankia sp. EAN1pec, R. eutropha*,

S. erythraea*, S. avermitilis*

C Frankia sp. CcI3, Frankia sp. KB5,

Frankia sp. UGL011102, UGL140104

# 2 A Frankia sp. EAN1pec, Anaeromyxobacter sp.*

D. ethenogenes*, C. hydrogenoformans*

B F. alni ACN14a, Frankia sp. CcI3,

Frankia sp. BCU110501

C Frankia sp. KB5, Frankia sp. CpI1,

. F. alni AvCI1 and ArI3 .

Table 4. Summary of the phylogenetic relationships between the large subunits (HupL)

of hydrogenase synton #1 (and #2) of various Frankia strains (also in comparison with

hydrogenases of other organisms). *Non-Frankia bacteria.

Alnus rubra) formed another group (Table 4). The Frankia strains which grouped

together might probably have related ancestors. Interestingly, the structural subunits of

both hydrogenase syntons #1 and #2 of Frankia sp. EAN1pec appear to be more closely

related to the hydrogenases of non-Frankia bacteria than they are to hydrogenases of

Frankia and thus it may have acquired its hydrogenase in a different way from the

Frankia strains in the two subgroups. We were unable to establish a connection between

the phylogenetic relationships of Frankia uptake hydrogenases and the geographical

distribution of Frankia strains or their hosts. However, a link between the biogeographic

history of the actinorhizal plants and the genome evolution of the bacterial symbionts

36

Page 36: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

has been proposed from a comparative study of the whole genomes of F. alni ACN14a,

Frankia sp. CcI3 and EAN1pec (Normand et al., 2007a).

The Hyp genes of Frankia hydrogenases are closer to those of bacteria than

archaea, and the Hyp genes of hydrogenases of F. alni ACN14a and Frankia sp. CcI3

are more closely related to each other than they are to those of Frankia sp. EAN1pec (for

both hydrogenase syntons #1 and #2). HypD1 and HypF1 (synton #1) and HypB2 and

HypE2 (synton #2) of F. alni ACN14a and Frankia sp. CcI3 are more strongly related to

those of non-Frankia bacteria than those of Frankia sp. EAN1pec. Frankia sp. CcI3 and

F. alni ACN14a have similar contents and orientation of genes in their uptake

hydrogenase synton #1 , while the syntons are both reduced and rearranged in Frankia

sp. EANpec, although Frankia EAN1pec has the largest genome (9.0 Mb), followed by

F. alni ACN14a (7.5 Mb) and Frankia sp. CcI3 (5.4 Mb) (Normand et al., 2007a; Fig.

3). Similarly, the content and orientation of genes in uptake hydrogenase synton #2 in F.

alni ACN14a and Frankia sp. CcI3 are very similar, but quite different in Frankia

EAN1pec. Frankia EAN1pec may have acquired its uptake hydrogenase by lateral gene

transfer from other non-Frankia bacteria in a different way from Frankia sp. CcI3 and F.

alni ACN14.

The regulation of uptake hydrogenases in Frankia

From the structural and phylogenetic studies outlined above, the uptake hydrogenase

syntons of Frankia are clearly not gene duplicates but different isozymes, which might

be required for hydrogen metabolism under different circumstances (Paper III). In

accordance with this hypothesis, there are indications that several microorganisms may

express different types of hydrogenases with differing specific functions under different

environmental conditions (Laurinavichene et al., 2002).

Hydrogenase gene expression in free-living vs. symbiotic condition: The transcript

levels of the structural subunit genes of uptake hydrogenase synton #1 (hupS1 and

hupL1) were higher than those of hydrogenase synton #2 (hupS2 and hupL2) in F. alni

ACN14a grown under free-living conditions. In contrast, the transcript levels of uptake

37

Page 37: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

hydrogenase synton #2 were higher than those of hydrogenase synton #1 under

symbiotic conditions, with observed hupL1:hupL2 expression ratios of 2:1 under free-

living conditions and 34:1 under symbiotic conditions. Therefore, synton #1 uptake

hydrogenases of Frankia are expressed more strongly under free-living conditions, and

synton #2 hydrogenases are mainly involved in symbiotic interactions (Paper III). It was

not possible to further confirm this result at a protein level since we have not as yet been

able to raise antibodies against the structural subunits of Frankia hydrogenases.

Ni-dependent regulation of uptake hydrogenases in Frankia: Nickel is an essential

micronutrient for many microorganisms, which is incorporated into at least four

microbial enzymes that participate in important reactions of hydrogen metabolism,

ureolysis, methane biogenesis, and acetogenesis (Hausinger, 1987). The presence of

nickel had previously been shown to have positive effects on the activity of uptake

hydrogenase in free-living Frankia strains (Sellstedt and Smith, 1990; Mattsson and

Sellstedt, 2002). In the studies underlying this thesis, the activity of the uptake

hydrogenases in Frankia UGL011301 and R43 were also found to be significantly

enhanced by the addition of nickel to the growth medium (Paper I). Since it is important

for microorganisms to maintain precise homeostasis of nickel ions in their cells, various

organisms have been shown to have evolved nickel-specific sensing and transport

systems (Eitinger and Mandrand-Berthelot, 2000), allowing them to take up nickel when

it is required, and to avoid doing so when it is not needed, via an inducible nickel-

resistance mechanism (Grass et al., 2003). Frankia may also have evolved a mechanism

to take up nickel when needed and avoid doing so when it is in excess, although there are

no data to support this hypothesis as yet

Thin cryosections of free-living Frankia strains treated with antiserum raised

against the hydrogenase of A. latus (the holoenzyme) showed that there were higher

degrees of hydrogenase transfer into the membranes of nickel-treated cells than in

nickel-free controls. The relative abundance of gold particles was much higher in

membranes when nickel had been added than in cells lacking nickel, where the gold

particles were localized mainly in the cytoplasm. As in various other organisms, the

38

Page 38: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

processing and correct positioning of the protein in the membrane of Frankia is essential

for the enzyme to be biologically active (Paper I).

Effects of nitrogenase and hydrogen on the uptake hydrogenase of Frankia: Frankia

sp. KB5 produces its uptake hydrogenase only when grown in nitrogen-free media.

Frankia sp. R43, on the other hand, produces its uptake hydrogenase when cells are

grown in both nitrogen-fixing and non-nitrogen-fixing conditions (Paper IV). Frankia

sp. R43, unlike other Frankia strains, can produce vescicles in both nitrogen-fixing and

non-nitrogen-fixing conditions (Fernando, 1991). A strong correlation between uptake

hydrogenase and nitrogenase activity in Frankia KB5 grown in nitrogen-fixing

conditions has previously been reported (Mattsson and Sellstedt, 2000). Some uptake

hydrogenases of Frankia may be produced independently of nitrogenase, but

dependently on the hydrogen produced as an inevitable byproduct of the nitrogen

fixation process. In accordance with this hypothesis, Frankia KB5 grown in non-

nitrogen-fixing conditions, but in the presence of exogenous hydrogen, can produce an

uptake hydrogenase (Mattsson and Sellstedt, 2000). In addition, Frankia sp. R43 may

have another type of uptake hydrogenase, which can be produced independently of both

nitrogenase and hydrogen. In contrast, uptake hydrogenase and nitrogenase synthesis are

coregulated in Rhizobium leguminosarum bv. viciae by the nitrogen fixation regulator

NifA (Brito et al., 1997) and two FnrN proteins (Gutiérrez et al., 1997). Coregulation of

hydrogenase and nitrogenase synthesis in R. capsulatus (by the RegB-RegA two-

component regulatory system) has also been reported (Dischert et al., 2000).

A hydrogen-evolving enzyme in Frankia

A broad range of Frankia strains grown in a medium without nitrogen (and without

nickel) were screened to investigate the presence of hydrogen-evolving hydrogenases in

the genus. The activity of the enzymes was measured as H2 production by gas

chromatography (Mattsson and Sellstedt, 2000). Ammonium chloride was added at the

start of induction of hydrogen evolution to block hydrogen production from the

39

Page 39: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

nitrogenase-catalyzed nitrogen-fixation process, and ARA measurements were taken to

check that all nitrogenase activity was eliminated.

Methyl viologen-mediated hydrogen evolution was recorded in only four Frankia

strains: F. alni UGL011101, UGL140102, Frankia sp. CcI3 and R43, originally isolated

from three different host plants. The highest rate of hydrogen evolution recorded was

obtained from Frankia UGL140102 (Paper IV). This is the first time, to our knowledge,

that evidence of methyl viologen-mediated hydrogen evolution in the actinomycete

Frankia has been published. The hydrogen recorded from Frankia sp. R43 did not

originate from an uptake hydrogenase acting in the reverse direction, since this protein

was not present in anaerobic conditions, as confirmed by the measurements of enzyme

activity and western blot analysis.

Molecular characterization of the hydrogen-evolving enzyme in Frankia

The presence of a hydrogen-evolving enzyme in Frankia was confirmed by Western

immunoblot analysis using antisera raised against HoxF of R. eutropha, which

recognized a 60 kDA protein in Frankia sp. R43 extracts (Paper IV).

Gel and peptide analysis: Frankia R43 proteins extracted from cells grown in anaerobic

conditions were electrophoretically separated on replicate 2-D gels. Protein spots of a

molecular weight of approx. 47 kDa identified by immunoblots were excised, digested

and analyzed using ESI-MS/MS Q-TOF. Short sequences in the resulting peptide

fingerprint showed nearly exact matches in searches of the NCBI protein database to

protein sequences of the bidirectional hydrogenase hoxH subunit of Anabaena siamensis

TISTR8012 (GenBank accession no. AAN65267). Thus, the hydrogen-evolving enzyme

in Frankia has strong similarity to this bidirectional cyanobacterial hydrogenase.

However, the metabolic function of the hydrogen-evolving hydrogenase in Frankia does

not appear to involve NAD-reduction, as proposed for the bidirectional hydrogenase in

cyanobacteria (Appel and Schulz, 1996), since no NAD-reducing activity was detected

in Frankia cells in which hydrogen evolution had been induced (Paper V). The

hydrogen-evolving hydrogenase in Frankia may act as an electron scavenger under

40

Page 40: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

anaerobic conditions, especially since Frankia is commonly found in

microaerophilic/anaerobic environments, e.g. in root nodules in its symbiotic form and in

river and lake sediments in its free-living form (Huss-Danell et al., 1997).

Localization of the hydrogen evolving enzyme in Frankia: Cryosectioning in

combination with immuno-gold labeling techniques using antibodies raised against

HoxH of the SH-hydrogenase HY of R. eutropha was performed to study the subcellular

localization of the hydrogenase in Frankia cells grown in nitrogen-limiting conditions

(and without nickel) for seven days and then kept anaerobically for 24 h (the conditions

used for the physiological measurements). A polypeptide was recognized by the

antibody in both hyphae and vesicles of Frankia sp. R43 (Paper IV) and Frankia sp.

UGL140102 (Paper V), the labeling being evenly distributed in these organelles,

indicating that the hydrogen-evolving hydrogenase is a soluble enzyme.

Regulation of the hydrogen-evolving enzyme in Frankia

Nitrogenase and the hydrogen-evolving hydrogenases of Frankia: Frankia sp. R43

showed methyl-viologen-mediated hydrogen evolution when grown in either nitrogen-

fixing or non-nitrogen fixing conditions. On the other hand, Frankia sp. KB5 showed

methyl-viologen-mediated hydrogen evolution only when grown in nitrogen-fixing

conditions (Paper III). Although it is premature to conclude that there is any correlation

between the expression of the nitrogenase and hydrogen-evolving enzymes, the

possibility that the nitrogenase enzyme affects the regulation of the hydrogen-evolving

enzyme directly or indirectly, at least in some Frankia strains, cannot be excluded. In

cyanobacteria, the level of activity of the bidirectional hydrogenase enzyme has been

found to be unaffected by exposing cells to hydrogen during their growth, and by the

addition of nitrogen to the growth medium, indicating that the expression this enzyme is

not dependent on, or even related to diazotrophic growth conditions, unlike their uptake

hydrogenase, the expression of which is linked to nitrogenase expression (Schütz et al.,

2004). The biosynthesis of nitrogenase is not essential for the biosynthesis of

bidirectional hydrogenase and hydrogen evolution in several unicellular strains (Howarth

41

Page 41: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

and Codd, 1985). Hydrogenases of cyanobacteria are expressed independently of

nitrogenase synthesis (Houchins, 1984).

Ni-dependent regulation of the hydrogen-evolving hydrogenases of Frankia: nickel

appears to play a role in the regulation of the hydrogen-evolving enzyme in Frankia.

Methyl viologen-mediated hydrogen evolution was recorded from F. alni UGL011103,

Frankia sp. UGL020602, UGL020603, and 013105 when grown in a medium without

nitrogen, but only in the presence of nickel (II) chloride (Paper V). These Frankia strains

did not display methyl viologen-mediated hydrogen evolution when grown in nitrogen-

fixing conditions without nickel (Paper I). Frankia may have different types of

hydrogen-evolving hydrogenases, or the hydrogen-evolving hydrogenases of Frankia

may at least be regulated differently in different Frankia strains. HypB genes encode

proteins that are highly conserved between different organisms and consist of at least

four domains (Robson, 2001). The presence of multiple histidinyl residues, which

characterizes the second domain of the proteins in several microorganisms like B.

japonicum, R. capsulatus, Azotobacter sp., B. leguminosarum (but which are known to

be missing in Archaea), suggests that this protein plays a role in binding divalent metals,

especially nickel (Rey et al., 1994; Robson, 2001). HypB, the nickel-binding GTPase, is

involved in nickel storage/sequestering and incorporation of nickel into the hydrogenase

in B. japonicum (Olson et al., 1997; Olson and Maier, 2000). The hydrogen sensor

hydrogenase HupUV, which regulates hydrogenase synthesis (e.g. in B. japonicum),

requires nickel to be active. The HypB2 of Frankia sp. CcI3 has seven histidinyl

residues in this domain while that of F. alni ACN14a has only one, and Frankia sp.

EAN1pec has none at all (Table 5). The mechanism whereby nickel regulates

hydrogenase transcription in Frankia is not known, but the available sequence

information indicates that, as in other organisms like B. japonicum, it may be via the

HypB, although this needs to be experimentally confirmed. HypB protein lacking the

nickel-binding polyhistidine region near the N terminus lacks the ability to store nickel,

but it is still able to bind a single nickel ion and also retains complete GTPase activity

(Rey et al., 1994).

42

Page 42: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

F. alni ACN14a*1 ------------------------------------------------MG 2 Frankia sp. CcI3*2 ----MGRFHPHPEGAHPHPEGAPHEYSGPHPPAGVS------------VG 34 Mycobacterium vanbaalenii3 ----MGRFHRHDDG-----TAHTHDHDG---SPSHD------------HG 26 Frankia sp. EAN1pec*4 ----MRRSGSPPSRCADLRAGWTSRRVGRPGR--LD--VPAGWSGGGLMC 42 Nodularia spumigena*5 MCVTCGCSDDAESTITNLETGEVEHNHHDHTHTLLDGTVISHSHNHDTQH 50 Methanosarcina mazei*6 -------------------------------------------------- Archaeoglobus fulgidus*7 -------------------------------------------------- F. alni ACN14a DHSGYRTGAE--RVEVLERILGENEKVARANRAAFDAAGVTVVNLMSAPG 50 Frankia sp. CcI3 DHAGYGTGPE--RVEVLERILGENERVALANRAAFDAAGVTVVNLMSAPG 82 Mycobacterium vanbaalenii DHSGYHTAAE--RVDVLEAIFSENDLRAAANRNAFEENGIRALNLMSSPG 74 Frankia sp. EAN1pec GTCGCEAETR--TLRLEMDVLARNEESADDNRAWLAARRASAVNLMSSPG 90 Nodularia spumigena EASQVHAKIHNTTISLEQDILAKNNLIAAQNRGWFKGRNILALNLMSSPG 100 Methanosarcina mazei -----MMFMLMHVIHMGHDVYKANDKIAEKNRKTLDKHGVFSVNVMGAIG 45 Archaeoglobus fulgidus ----------MHEYELNQDLLAENKRLAEKNREALRESGTVAVNIMGAIG 40 : : *. * ** : :*:*.: *

Table 5. Multiple sequence alignments of N-terminal sequence of the HypB proteins

from Frankia (hydrogenase synton #2), non-Frankia bacteria and Archaea (produced by

ClustalW). *1-*7 Protein_IDs: *1 = 111221273, *2 = 86739790, *3 = 120403348, *4 =

68233089 *5 = 119509235, *6 = 21228420 and *7 = 11498964. The Domain 2 of HypB

proteins of many Eubacteria are known to have multiple histidinylresidues that are known

to bind divalent metals, especially nickel.

Does Frankia have other hydrogenases?

Frankia may also produce Fe-only hydrogenases, since hydrogenases of Frankia

UGL011102 and Frankia KB5 were found to be immunologically related to the [Fe]-

hydrogenase of D. desulfuricans ATCC 7757. However, more experiments are required

to confirm this finding.

Conclusions

Frankia has at least three types of hydrogenases.

Uptake hydrogenases are common in Frankia and are probably Ni-Fe hydrogenases.

Frankia commonly has two uptake hydrogenase syntons, which are distinctively

separated in their genome. The structural, accessory and regulatory genes of these

43

Page 43: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

hydrogenases syntons are organised tightly together. These hydrogenase syntons differ

in many ways.

Hydrogenase synton #1 and #2 in a Frankia are phylogenetically divergent and

hydrogenase synton #1 is probably ancestral among the actinobacteria.

The hydrogenases genes of F. alni ACN14a and Frankia sp. CcI3 are closely related but

relatively distant from those of Frankia sp. EAN1pec, which was more related to the

hydrogenases of non-Frankia bacteria than Frankia. The tree topology is indicative of

probable gene transfer to or from ancestral organisms that occurred before the

emergence of Frankia. All of the available evidence points to hydrogenase gene

duplication having occurred long before development of the three Frankia lineages.

Uptake hydrogenase synton #1 of Frankia is expressed more strongly under free-living

conditions but hydrogenase synton #2 is mainly involved in symbiotic interactions.

Nickel enhances the degree of hydrogenase transfer into the membranes and the activity

of uptake hydrogenases in Frankia. The processing and correct positioning of the protein

in the membrane of Frankia is essential for the enzyme to be biologically active.

Some Frankia strains have a hydrogen-evolving enzyme. This hydrogenase is a soluble

enzyme as it is localized in both hyphae and vesicles, and is related to the bidirectional

cyanobacterial hydrogenases.

Unlike other Frankia strains, Frankia sp. R43 showed a methyl-viologen-mediated

hydrogen evolution and uptake hydrogenase activity in both nitrogen-fixing and non-

nitrogen-fixing conditions.

Nickel appears to play a role in the regulation of the hydrogen-evolving enzyme in

Frankia as some Frankia strains showed activity of this enzyme only in the presence of

this metal. Frankia may have different types of hydrogen-evolving hydrogenases, or the

44

Page 44: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

hydrogen-evolving hydrogenases of Frankia may at least be regulated differently in

different Frankia strains.

Hydrogen-evolving enzyme of Frankia evolves a considerable amount of hydrogen that

can be used as a clean energy source.

Future perspectives

It would be interesting to further study:

• The activities of uptake hydrogenases synton #1 and #2 of F. alni ACN14a in

symbiotic vs. free-living conditions to in order to confirm the present result of

their differential expression under these conditions. It would also be interesting to

see if this difference also exists in the uptake hydrogenase synton #1 and #2 of

other Frankia strains.

• The effect of nickel and selenium on uptake hydrogenase synton #1 and #2 of

Frankia at both mRNA and protein levels.

• The Fe only hydrogenases Frankia strains.

• The effect of different physiological conditions on the activity of the hydrogen-

evolving hydrogenases in Frankia. This helps to know the optimum condition in

which maximum biological hydrogen production can be obtained from Frankia.

45

Page 45: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Frankia – bakterien som pruducerar både kväve gödsel och vätgas!

Alla 18 Frankia stammar som isolerats från tio aktinorhiza värdväxter härstammande

från olika delar av världen, hade det väte-förbrukande enzymet hydrogenas. Tillsättning

av nickel till odlingsmediet ökade enzymets aktivitet och mängd i membran, vilket tyder

på att nickel behövs för ett aktivt enzym och att enzymet sannolikt är ett Ni-Fe

hydrogenas.

Karakterisering av tre Frankia genom v

isade närvaro av två helt skilda hydrogenas kluster. De två hydrogenasen är även vanliga

hos andra Frankia stammar. De strukturella, reglerande och accessoriska generna i

hydrogenas kluster#1 och #2 ligger intill varandra, men har olika struktur. Medans

hydrogenas kluster #1 (eller kluster #2) av F. alni ACN14a och Frankia sp. CcI3 har

likande gen sammansättning, innehåll och orientering har Frankia sp. EAN1pec minskat

antal gener och är annorlunda organiserat. Hydrogenas från Frankia sp. ACN14a och

CcI3 är fylogenetiskt mer relaterad till varandra än till Frankia sp. EAN1pec, som i sin

tur är mer relaterad till en icke-Frankia bakterie. Fylogenetiska och topologiska studier

tyder på en sannolik genöverföring till eller från Frankia långt innan Frankias

uppkomst. De väte-förbrukande hydrogenasets kluster #1 är mer uttryckt i frilevande

Frankia medans hydrogenas kluster #2 är huvudsakligen involverad i rotknölar. Detta

hydrogenas kan spela en betydande roll för att öka effektiviteten av kväve-fixeringen i

rotknölar hos värdväxter med Frankia och därmed kan behoven av miljöovänligt och

dyrt gödningsmedel minskas.

De väte-producerande hydrogenaset återfanns endast hos fyra Frankia stammar,

F. alni UGL011101, UGL140102, Frankia sp. CcI3 och R43. Efter tillsättning av nickel

visade även F. alni UGL011103, Frankia sp. UGL020602, UGL020603, och 013105

aktivitet. Nickel bidrog också här till en ökning, vilket indikerar att Frankia möjligen

har olika typer av väte-producerande hydrogenas, eller att enzymet regleras på olika sätt

i olika Frankia stammar. Det faktum att Frankia producerar väte blev nyligen känt.

Därför är molekylärbiologiska kunskaper om hydrogenaser av yttersta vikt för att

optimera ett system som kan bli en biologisk producent av väte.

46

Page 46: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Acknowledgements

First and foremost, praise and thanks goes to my heavenly father, who loved me and gave

me His only begotton Son, my Lord Jesus Christ, and in Him, everything else. May His

Name be glorified for ever and ever, Amen!

I am privileged to do my PhD studies at Fys-Bot, which is for sure the nicest working

environment I have ever experienced. I am very thankful to all senior scientists, PhD

students, technical staff, post-docs for contributing to this work, directly or indirectly.

Special thanks to:

My Parents – you didn’t have a lot for your self, but you gave me all you have. You

believed in me, you encouraged me, you loved me. Thank you very much.

My better half – Genni, you are a special gift to me! Thank you for being patient when

we lived apart. Thank you for your love, understanding, encouragement and prayers.

Anita Sellstedt – my supervisor, for introducing me to molecular biology and laboratory

work, for providing me with all I needed for my study (for allowing me to attend

international conference etc), for the courage you gave me by believing in me. You have

been very understanding, especially in times of need, thank you! Thank you for helping

me with, among many other things, correcting the thesis.

Berhanu, Barbro, Rut and Hanna – Thank you for kindness. You have been a real

blessing to our life. Thank you for your concern, advice and prayers. You have a special

place in our heart.

Franscesco (Sir) – for being very nice to me. Thanks for the company, all the story we

shared together, for the ideas at meetings. Marie – thanks for your kindness and concern.

You have been very helpful. You are so organized, thank you for being a good example.

47

Page 47: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Anasuya, Prabha, Lars, Mats-Jerry, João, Jenny, Catarina and others - my Lab mates (of

the past), thank you for helpful ideas and your contribution. You have been so nice.

Stefan Jansson (Prof) and Per Gardeström (Prof) – my reference group, thank you for

your valuable comments.

All PhD students (of the last 5 years) – Jacob, Stefan, Andreas, Henrik, Maribel, Junko,

Charlene and many others – for helpful interaction and contribution to my work.

Philippe Normand (Prof) – for being available for advice. You have been very helpful.

John Blackwell – for correcting my English.

Slim, Inger, Monika, Siv, Karin, Brit-Marie, Gunilla, Ulrika, Eva, Susie, Rupali, Per-

Ingvar, Leszek, Laszlo, Catherine, Roland, Paavo, Lars, Janne, Hannele, Benedicte,

Simon, Jan, Thomas, Frank, Arsenio, Estelle, Luis and others – you have been very

helpful in the many ways I needed you, thank you!

Ale, Tesfu, Ben, Kalu, the king, Bezi – for your kindness and support; Wube, Aseged,

Abebaw, Chuni, Baby – for you kindness and support; Bekele, Tsehay, Betty, Eyerus,

Hanna – for your love and concern and prayers.

The Assosa community – for the advice, encouragement and fun.

Emmanuelgruppen - Tsehay and Mike; Saba and Haile; Tenagne and Jhoni; Dawit and

Martha; Misgina and Yordi; Mike; Firewoyni; all the small lovely kids. Thank you for

the great fellowship. Misgina, thanks for the hug! Yeshewas, you have been very helpful.

Fremi – thank you for correctiong my Swedish, stories we shared, for being a nice friend.

Haidy and Mattias; Per-Olov and Habibe - you made our life in Umeå easier. Thank you

for your advice, generosity and friendship.

Kemiförrådet, Vaktmästeriet KBC and Caelum (catering) staffs – for the help and fun.

48

Page 48: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

References

Adams MWW (1990) The structure and mechanism of iron hydrogenases. Biochim

Biophys Acta 1020: 115-45.

Adams MWW, Mortenson LE, Chen J-S (1981) Hydrogenase. Biochim Biophys Acta

594: 105-76.

Adams MWW and Stiefel EI (1998) Biological hydrogen production: Not so elementary.

Science 282: 1842-43.

Afting C, Hochheimer A and Thauer RK (1998) Function of H2-forming

methylenetetrahydrometha-nopterin dehydrogenase, a metal free hydrogenase in

methanogenic Archaea growing on H2 and CO2. Arch Microbiol 169: 206-10.

Appel J, Phunpruch S, Steinmüller K and Schulz R (2000) The bidirectional

hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during

photosynthesis. Arch Microbiol 173: 333-38.

Appel J and Schulz R (1996) Sequence analysis of an operon of a NAD(P)-reducing

nickel hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 gives

additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase

(complex I). Biochim Biophys Acta 1298 (2): 141-47.

Baker DD (1987) Relationships among pure cultured strains of Frankia based on host

specificity. Physiol Plantarum 70: 245-48.

Baker D and Torrey JG (1980) Characterization of an effective actinorhizal

microsymbiont, Frankia sp. AvCI1 (Actinomycetales). Can J Microbiol 26: 1066-71.

49

Page 49: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Benson DR, Vanden Heuvel BD and Potter D (2004) Actinorhizal symbioses: Diversity

and biogeography. In Gillings M (ed) Plant microbiology. BIOS Scientific Publishers

Ltd., Oxford.

Benson DR and Silvester WB (1993) Biology of Frankia Strains, actinomycete

symbionts of actinorhizal plants. Microbiol Rev 57: 293-319.

Berghofer Y, Agha-Amiri K and Klein A (1994) Selenium is involved in the negative

regulation of the expression of selenium-free [NiFe] hydrogenases in Methanococcus

voltae. Mol Gen Genet 242: 369-73.

Berry A and Torrey JG (1979) Isolation and characterization in vivo and in vitro of an

actinomycetous endophyte from Alnus rubra Bong, p. 69–83. In Gordon JC, Wheeler CT

and Perry DA (eds), Symbiotic nitrogen fixation in the management of temperate forests.

Forest Research Laboratory, Oregon State University, Corvallis.

Binkley D, Cromack K Jr and Baker DD (1994) N fixation by red alder: biology, rates

and controls, p. 57–72. In Hibbs D, DeBell D and Tarrant R (eds), The biology and

management of red alder. Oregon State University Press, Corvallis.

Black LK, Fu C and Maier RJ (1994) Sequence and characterization of hupU and hupV

genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex

involved in hydrogenase expression. J Bacteriol 176: 7102-06.

Brito B, Martínez M, Fernández D, Rey L, Cabrera E, Palacios JM, Imperial J and Ruiz-

Argüeso T (1997) Hydrogenase genes from Rhizobium leguminosarum bv. viciae are

controlled by the nitrogen fixation regulatory protein NifA. Proc Natl Acad Sci USA 94:

6019-24.

Callaham D, DelTredici P and Torrey JG (1978) Isolation and cultivation in vitro of the

actinomycete causing root nodulation in Comptonia. Science 199: 899–902.

50

Page 50: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Cammack R (2001) Hydrogenases and their activities, p. 9-32. In Cammack R and

Florence KY (eds), Hydrogen as fuel: Learning from nature. London: Taylor & Francis.

Chaia E (1998) Isolation of an effective strain of Frankia from nodules of Discaria

trinervis (Rhamnaceae) Plant and Soil 205: 99-102.

Chaudhary AH and Mirza MS (1987) Isolation and characterization of Frankia from

nodules of actinorhizal plants of Pakistan. Physiol Plantarum 70: 255-58.

Clawson ML, Bourret A and Benson DR (2004) Assessing the phylogeny of Frankia-

actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and

glutamine synthetase gene sequences. Mol Phylogenet Evol 31: 131-38.

Diem HG and Dommergues YR (1990) Current and potential uses and management of

Casuarinaceae in the tropics and subtropics, p. 317-342. In Schwintzer CR and

Tjepkema JD (eds), The biology of Frankia and actinorhizal plants. Academic Press,

Inc. San Diego, California.

Dischert SW, Colbeau A and Bauer CE (2000) Expression of Uptake Hydrogenase and

Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA

Two-Component Regulatory System. J Bacteriol 182: 2831-37.

Dixon ROD (1972) Hydrogenase in legume root nodule bacteroids: occurrence and

properties. Arch Microbiol 85: 193-201.

Dixon ROD (1976) Hydrogenases and efficiency of nitrogen fixation in aerobes. Nature

263: 173.

Eitinger T and Mandrand-Berthelot M-A (2000) Nickel transport systems in

microorganisms. Arch Microbiol 173: 1-9.

51

Page 51: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Elsen, S., Colbeau, A., Chabert, J. & Vignais, P. M. (1996) The hupTUV operon is

involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. J

Bacteriol 178: 5174-81

Evans HJ, Koch B and Klucas R (1972) Preparation of nitrogenase from nodules and

separation into components. Methods Enzymol 24: 470-76.

Fernando Tavares (1991). Género Frankia (Actinomycetales) Aspectos morfológicos e

ultraestruturais. Msc thesis.

Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap.

Evolution 39: 783-91.

Filipiak M, Hagen WR and Veeger C (1989) Hydrodynamic, structural and magnetic

properties of Megasphaera elsdenii Fe hydrogenase reinvestigated. Eur J Biochem 185:

547-53.

Friedrich B, Heine E, Finck A and Friedrich CG (1980) Nickel requirement for active

hydrogenase formation in Alcaligenes eutrophus. J Bacteriol 145 (3): 1144-49.

Friedrich B and Schwartz E (1993) Molecular biology of hydrogen utilization in aerobic

chemolithotrophs. Annu Rev Microbiol 47: 351-83.

Friedrich B, Vignaise PM, Lenz O and Colbeau A (2001) Regulation of hydrogenase

gene expression, p. 33-56. In Cammack R and Florence KY (eds), Hydrogen as fuel:

Learning from nature. London: Taylor & Francis.

Garcin E, Vernede X, Hatchikian EC, Volbeda A, Frey M and Fontecilla-Camps JC

(1999) The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the

activated catalytic center. Structure 7: 557-66.

52

Page 52: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Gaston KJ and Spicer JI (2004) Biodiversity: an introduction. Oxford: Blackwell

Publishing, 2nd ed., ISBN 1-4051-1857-1

Grass G, Fan B, Rosen BP, Lemke K, Schlegel HG and Rensing V (2001) NreB from

Achromobacter xylosoxidans 31A is a nickel induced transporter conferring nickel

resistance. J Bacteriol 183: 2803-07.

Gutiérrez D, Hernando Y, Palacios JM, Imperial J and Ruiz-Argüeso T (1997) FnrN

controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium

leguminosarum biovar viciae UPM791. J Bacteriol 179: 5264-70.

Hanus F, Maier RJ and Evans H (1979) Autotrophic growth of H2-uptake positive strains

of Bradyrhizobium japonicum in an atmosphere supplied with hydrogen gas. Proc Natl

Acad Sci USA 76: 1788-92.

Hausinger RP (1987) Nickel Utilization by Microorganisms. Microbiol Rev 51: 22-42.

Higuchi Y, Yagi T and Yasouka N (1997) Unusual ligand structure in Ni-Fe active

center and an additional Mg site in hydrogenase revealed by high resolution X ray

structure analysis. Structure 5: 1671-80.

Houchins JP (1984) The physiology and biochemistry of hydrogen metabolism in

cyanobacteria. Biochim Biophys Acta 768: 227-55.

Howarth DC and Codd GA (1985) The uptake and production of molecular hydrogen by

unicellular cyanobacteria. J Gen Microbiol 131: 1561-69.

Huss-Danell K and Bergman B (1990) Nitrogenase in Frankia from Root Nodules of

Alnus incana (L.) Moench: immunolocalization of the Fe- and MoFe-proteins during

vesicle differentiation. New Phytologist 116: 443-455.

53

Page 53: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Huss-Danell K, Uliassi D and Renberg I (1997) River and lake sediments as sources of

infective Frankia (Alnus). Plant and Soil 197(1): 35-9.

Jeong SC, Ritchie NJ and Myrold DD (1999) Molecular Phylogenies of Plants and

Frankia Support Multiple Origins of Actinorhizal Symbioses. Mol Phylogenet Evol 13

(3): 493-503.

Karube I, Matsunaga, Tsuni S and Suzuki S (1976) Continuous hydrogen production by

immobilized whole cell of Clostridium butyricum. Biochem Biophys Acta 44: 338-45.

Kennedy C and Toukdarian A (1987) Genetics of azotobacters: applications to nitrogen

fixation and related aspects of metabolism. Annu Rev Microbiol 41: 227-58.

Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions

through comparative studies of nucleotide sequences. J Mol Evol (16): 111-20.

Kleihues L, Lenz O, Bernhard M, Buhrke T and Friedrich B (2000) The H2 Sensor of

Ralstonia eutropha Is a Member of the Subclass of Regulatory [NiFe] Hydrogenases. J

Bacteriol 182: 2716-24.

Kovács AT, Rákhely G, Balogh J, Maróti G, Fülöp A and Kovács KL (2005) Anaerobic

regulation of hydrogenase transcription in different bacteria. Biochem Soc Transact 33:

36-38.

Kovács KL and Rákhely G (2007) Thiocapsa roseopersicina Hydrogenases: Why There

Are So Many and What Do They Do? Lecture Abstract. The 8th International

Hydrogenase Conference, August 5-10, Breckenridge, Colorado.

Kovács KL, Seefeldt LC, Tigyi G, Doyle CM, Mortenson LE and Arp DJ (1989)

Immunological relationship among hydrogenases. J Bacteriol 171: 430-35.

54

Page 54: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Laurinavichene TV, Zorin NA, Tsygankov AA (2002) Effect of redox potential on

activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli. Arch Microbiol 178(6):

437-42.

Lalonde M, Calvert HE and Pine S (1981) Isolation and use of Frankia strains in

actinorhizae formation, p. 296-99. In Gibson AH and Newton WE (eds), Current

Perspectives in Nitrogen Fixation. Australian Academy of Science, Canberra.

Lalonde M, Simon L, Bousquet J and Séguin A (1988) Advances in the taxonomy of

Frankia: recognition of species alni and elaeagni and novel subspecies pommerii and

vandijkii, p. 671-80. In Bothe H, Bruijn FJ and Newton WE (eds), Nitrogen Fixation:

Hundred Years After. Fischer, Stuttgart.

Lindmark DG and Muller M (1973) Hydrogenosome, a cytoplasmic organelle of the

anaerobic flagellate, Trichomonas foetus, and its role in pyruvate metabolism. J Biol

Chem 248: 7724-28.

Lumini E, Bosco M and Fernandez MP (1996) PCR-RFLP and total DNA homology

revealed three related genomic species among broad-host-range Frankia strains. FEMS

Microbiol Ecol 21(4): 303-11.

Lyon EJ, Shima S, Buurman G, Chowdhuri S, Batschauer A, Steinbach K, Thauer RK

(2004) UV-A/blue-light inactivation of the ‘metal-free’ hydrogenase (Hmd) from

methanogenic archaea. Eur J Biochem 271: 195-204.

Matias PM, Soares CM, Saraiva LM, Coelho R, Morais J, Gall JL and Carrondo MA

(2001) [NiFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 27774: Gene

sequencing, three-dimensional structure determination and refinement at 1.8 Å and

modeling studies of its interaction with the tetrahaem cytochrome c3. J Biol Inorg Chem

6: 63-81.

Mattsson U (2001) Hydrogenases in Frankia. Phd thesis. ISBN 91-7191-942-2

55

Page 55: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Mattsson U, Johansson L, Sandström G and Sellstedt A (2001) Frankia sp. KB5

possesses a hydrogenase immunologically related to membrane-bound [NiFe]-

hydrogenases. Curr Microbiol 42: 438-41.

Mattsson U and Sellstedt A (2000) Hydrogenase in Frankia sp. KB5: expression of and

relation to nitrogenase. Can J Microbiol 46: 1091-95.

Mattsson U and Sellstedt A (2002) Nickel affects activity more than expression of

hydrogenase protein in Frankia. Curr Microbiol 44: 88-93.

Meesters TM (1987) Localization of nitrogenase in vesicles of Frankia sp. Cc1.17 by

immunogoldlabelling on ultrathin cryosections. Arch Microbiol 146: 327-31.

Melis A, Zhang L, Forestier M, Ghirardi ML and Seibert M (2000) Sustained

photobiological hydrogen gas production upon reversible inactivation of oxygen

evolution in the green alga Chlamydomonas reinhardtii. Plant Physiology 122(1): 127-

36.

Mertens R and Liese A (2004) Biotechnological applications of hydrogenases. Curr

Opin Biotechnol 15(4): 343-48.

Montet Y, Amara P, Volbeda A, Vernede X, Hatchikian EC, Field MJ, Frey M and

FontecillaCamps JC (1997) Gas access to the active site of Ni-Fe hydrogenases probed

by Xray crystallography and molecular dynamics. Nature Struct Biol 4: 523-26.

Monz CA and Schwintzer CR (1989) The physiology of spore-negative and spore-

positive nodules of Myrica gale. Plant and Soil 118: 75-87.

Nazaret S, Cournoyer B, Normand P, and Simonet P (1991) Phylogenetic relationships

among Frankia genomic species determined by use of amplified 16S rDNA sequences. J

Bacteriol 173: 4072-78.

56

Page 56: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Nicolet Y, Cavazza C and Fontecilla-Camps JC (2002) Fe-only hydrogenases: structure,

function and evolution. J Inorg Biochem 91: 1-8.

Nicolet Y, Piras C, Legrand P, Hatchikian CE and Fontecilla-Camps JC (1999)

Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination

to an active site Fe binuclear center. Structure Fold Des 7(1): 13-23.

Normand P and Lalonde M (1982) Evaluation of Frankia strains isolated from

provenances of two Alnus species. Can J Microbiol 28: 1133-42.

Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, Bagnarol E, Bassi CA, Berry

AM, Bickhart DM, Choisne N, Couloux A, Cournoyer B, Cruveiller S, Daubin V,

Demange N, Francino MP, Goltsman E, Huang Y, Kopp OR, Labarre L, Lapidus A,

Lavire C, Marechal J, Martinez M, Mastronunzio JE, Mullin BC, Niemann J, Pujic P,

Rawnsley T, Rouy Z, Schenowitz C, Sellstedt A, Tavares F, Tomkins JP, Vallenet D,

Valverde C, Wall LG, Wang Y, Medigue C and Benson DR. (2007a) Genome

characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host

plant biogeography. Genome Res 17: 7-15.

Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J, Evtushenko L and

Misra AK (1996) Molecular Phylogeny of the Genus Frankia and Related Genera and

Emendation of Family Frankiaceae. Int J Syst Bacteriol 46: 1-9.

Normand P, Queiroux C, Tisa LS, Benson DR, Rouy Z, Cruveiller S and Médigue C

(2007b) Exploring the genomes of Frankia. Physiol Plantarum 130: 331-43.

Olson JW, Fu C and Maier RJ (1997) The HypB protein from Bradyrhizobium

japonicum can store nickel and is required for the nickel-dependent transcriptional

regulation of hydrogenase. Mol Microbiol 24: 119-28.

57

Page 57: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Olson JW and Maier RJ (2000) Dual roles of Bradyrhizobium japonicum nickelin

protein in nickel storage and GTP-dependent Ni mobilization. J Bacteriol 182: 1702-05.

Page RDM (1996) TREE VIEW: an application to display phylogenetic trees on

personal computers. Comput Appl Biosci 12: 357-58.

Parsons R, Silvester WB, Harris S, Gruijters WTM and Bullivant S (1987) Frankia

Vesicles Provide Inducible and Absolute Oxygen Protection for Nitrogenase. Plant

Physiol 83(4): 728-31.

Peters JW, Lanzilotta WN, Lemon BJ and Seefeldt LC (1998) The X-ray Crystal

Structure of the Fe Hydrogenase (CpI) from Clostridium pasteurianum to 1.8 A

Resolution. Science 282: 1853-58.

Pfaffi MW, Horgan GW and Dempfle L (2002) Relative expression software tool

(REST©) for group-wise comparison and statistical analysis of relative expression results

in real-time PCR. Nucleic Acids Research 30(9): e36.

Przybyla AE, Robbins J, Menon N and Peck HD Jr (1992) Structure-function

relationships among the nickel-containing hydrogenases. FEMS Microbiol Rev 88: 109-

36.

Rey L, Imperial J, Palacios J-M, and Ruiz-Argüeso T (1994) Purification of Rhizobium

leguminosarum HypB, a Nickel-Binding Protein Required for Hydrogenase Synthesis. J

Bacteriol 176: 6066-73.

Robson R (2001) Biodiversity of hydrogenases, p. 73-92. In Cammack R and Florence

KY (eds), Hydrogen as fuel: Learning from nature. London: Taylor & Francis.

Saitou N and Nei M (1987) The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol Biol Evol 4: 406-25.

58

Page 58: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Sayed WF, Wheeler CT, Zahran HH and Shoreit AAM (1997) The effect of temperature

and soil moisture on the survival and symbiotic effectivity of Frankia. Biol Fertil Soils

25: 349-53.

Schmitz O, Boison G, Salzmann H, Bothe H, Schütz K, Wang S-h and Happe T (2002)

HoxE-a subunit specific for the pentameric bidirectional hydrogenase complex

(HoxEFUYH) of cyanobacteria. Biochim Biophys Acta 1554: 66-74.

Schubert KR and Evans HJ (1976) Hydrogen evolution: a major factor affecting the

efficiency of nitrogen fixation in nodulated symbionts. Proc Natl Acad Sci USA 73:

1207-11.

Schwencke J (2001) Advances in Actinorhizal Symbiosis: Host Plant-Frankia

Interactions, Biology, and Applications in Arid Land Reclamation. Arid Land Res

Manag 15: 285-327.

Schütz K, Happe T, Troshina O, Lindblad P, Leitão E, Oliveira P and Tamagnini P

(2004) Cyanobacterial H2 production - a comparative analysis. Planta 218: 350-59.

Sellstedt A (1989) Occurrence and activity of hydrogenase in symbiotic Frankia from

field-collected Alnus incana. Physiol Plantarum 75: 304-8.

Sellstedt A, Huss-Danell K and Ahlqvist A-S (1986) Nitrogen fixation and biomass

production in symbiosis between Alnus incana and Frankia strains with different

hydrogenase metabolism. Physiol Plantarum 66: 99-107.

Sellstedt A and Lindblad P (1990) Activities, Occurrence, and Localization of

Hydrogenase in Free-Living and Symbiotic Frankia. Plant Physiol 92: 809-15.

59

Page 59: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Sellstedt A, Reddell P and Rosbrook P (1991) The Occurrence of Hemoglobin and

Hydrogenase in Nodules of 12 Casuarina-Frankia Symbiotic Associations. Physiol

Plantarum 82: 458-64.

Sellstedt A, Rosbrook PA, Kang L and Reddell P (1994) Effect of Carbon Source on

Growth, Nitrogenase and Uptake Hydrogenase Activities of Frankia Isolates from

Casuarina sp. Plant and Soil 158: 63-8.

Sellstedt A and Smith GD (1990) Nickel Is Essential for Active Hydrogenase in Free-

Living Frankia Isolated from Casuarina. FEMS Microbiol Lett 70: 137-40.

Sellstedt A, Wullings B, Nyström U and Gustafsson P (1992) Identifcation of

Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary

primers. FEMS Microbiol Lett 93: 1-6.

Silvester WB (1976) Ecological and economic significance of the non-legume

symbiosis, p. 489-506. In W. E. Newton and C. J. Nyman (eds), Proceedings of the First

International Symposium on Nitrogen Fixation. Washington University Press, Pullman.

Silvester WB (1977) Dinitrogen fixation by plant associations excluding legumes. In

Hardy R and Silvester W (eds), A treatise of dinitrogen fixation, Section IV: Agronomy

and Ecology, p. 141-90, John Wiley and Sons, New York.

Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wünschiers R and Lindblad P (2002)

Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev

66(1): 1-20.

Tamagnini P, Troshina O, Oxelfelt F, Salema R and Lindblad P (1997) Hydrogenases in

Nostoc PCC 73102, a strain lacking a bidirectional enzyme. Appl Environ Microbiol 63:

1801-07.

60

Page 60: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Tard C, Liu X, Ibrahim SK, Bruschi M, Gioia LD, Davies SC, Yang X, Wang L-S,

Sawers G and Pickett CJ (2005) Synthesis of the H-cluster framework of iron-only

hydrogenase. Nature 433: 610-13.

Thauer RK, Klein AR and Hartmann GC (1996) Reactions with Molecular Hydrogen in

Microorganisms: Evidence for a Purely Organic Hydrogenation Catalyst. Chem Rev 96:

3031-42.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F and Higgins DG (1997) The

CLUSTAL X windows interface: fexible strategies for multiple sequence alignment

aided by quality analysis tools. Nucleic Acids Res 25: 4876-82.

Torres V, Ballesteros A and Fernandez VM (1986) Expression of hydrogenase activity

in Barley (Hordeum vulgare L.) after anaerobic stress. Arch Biochem Biophys 245: 174-

78.

Troshina O, Serebryakova LT, Sheremetieva ME and Lindblad P (2002) Production of

H2 by the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 during

fermentation. Int J Hydrogen Energy 27: 1283-9.

Ruiz-Argüeso T, Imperial J and Palacios JM (2000) Uptake hydrogenases in root nodule

bacteria. In Triplett EW (ed), Prokaryotic nitrogen fixation: A model system for the

analysis of a biological process. Madison, WI: Horizon Scientific Press.

Van Soom C, Verreth C, Sampaio MJ and Vanderleyden J (1993) Identification of a

potential transcriptional regulator of the hydrogenase activity in free-living

Bradyrhizobium japonicum strains. Mol Gen Genet 239: 235-40.

Velázquez E, Cervantes E, Igual J M, Peix A, Mateos PF, Benamar S, Moiroud

A,Wheeler CT, Dawson JO, Labeda D, Rodríguez-Barrueco C and Martínez-Molina E

61

Page 61: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

(1998) Analysis of LMW RNA profiles of Frankia strains by staircase Electrophoresis.

Syst Appl Microbiol 21: 539-45.

Verhagen M-F, O'Rourke T and Adams MWW (1999) The hyperthermophilic

bacterium, Thermotoga maritima, contains an unusually complex iron-hydrogenase:

amino acid sequence analyses versus biochemical Characterization. Biochim Biophys

Acta 1412: 212-29.

Vignais P M, Billoud B and Meyer J (2001) Classification and phylogeny of

hydrogenases. FEMS Microbiol Rev 25: 455-501.

Vignais PM and Colbeau A (2004) Molecular Biology of Microbial Hydrogenases. Curr

Issues Mol Biol 6: 159-88.

Vignais PM and Toussaint B (1994) Molecular biology of membrane-bound H2 uptake

hydrogenases. Arch Microbiol 161: 1-10.

Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M and Fontecilla-Camps JC

(1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature

373: 580-87.

Voordouw G (1992) Evolution of hydrogenase genes. Adv Inorg Chem 38: 397-422.

Wall LG (2000) The actinorhizal symbiosis. J Plant Growth Regul 19: 167-82.

Wheeler CT, McEwan NR, Sellstedt A and Sandström G (1998) Application of

molecular techniques to ecological studies of symbioses in actinorhizal plants, p. 41-63.

In Warma A (ed), Mycorrhiza Manual, Springer-Verlag, Berlin.

Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis T and Mann M

(1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-

electrospray mass spectrometry. Nature 379: 466-9.

62

Page 62: The Biodiversity of Hydrogenases in Frankiaumu.diva-portal.org/smash/get/diva2:141056/FULLTEXT01.pdf · The Biodiversity of Hydrogenases in Frankia Characterization, regulation and

Wu L-F and Mandrand MA (1993) Microbial hydrogenases: primary structure,

classification, signatures and phylogeny. FEMS Microbiol Rev 10: 243-70.

Zhang Z, Lopez MF and Torrey JG (1984) A comparison of cultural characteristics and

infectivity of Frankia isolates from root nodules of Casuarina species. Plant and Soil

78: 79-90.

63


Recommended