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Coastal margin science: Microbiology Microbial Diversity and Microbial-mediated Activities. Peter Zuber. 1. Coastal margin science: Microbiology. Identify areas of microbial ‘productivity’ related to prevalent events or gradients in PNW coastal margin - PowerPoint PPT Presentation
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Coastal margin science: Microbiology Microbial Diversity and Microbial-mediated Activities
Peter Zuber
“CMOP: Transforming Ocean Exploration”
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Coastal margin science: Microbiology
Microorganisms
Ecosystem & human health
nationalgeographic.com
Human activity& climate
Physical circulation and transport
Chemicalstressors
Biological mediation
1. Identify areas of microbial ‘productivity’ related to prevalent events or gradients in PNW coastal margin
2. Developing new technologies to regularly and accurately monitor microbial-mediated activities
“CMOP: Transforming Ocean Exploration”
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Events & high gradient regions
- Coastal hypoxia
- Upwelling/downwelling regimes
- Climate variability
- Estuary/plume fronts
- River-to-ocean salinity gradients
- Estuarine turbidity maxima
- Steep vertical gradients in physical, biological & chemical propertiesNOAA web site
Sea surface temperatureImage from PISCO (OSU)
Fish mortality in July 2002 Grantham et al. 2004 Nature
CORIE forecast
Estuarine Turbidity MaximumEnhanced microbial productivity
“CMOP: Transforming Ocean Exploration”
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River, estuary, plume & ocean
Estuary & plumeDynamic environment
Forecast of bottom water salinity
Salinity (PSU)Frey et al 1984
Low phytoplankton cell counts in the Columbia river
estuary
Particulate Organic Carbon Inputs
Detrital particulateorganic carbon146,000 Mt/y
phytoplankton61,000 Mt/y
Primaryproduction
17,000 Mt/y
?
Salinity (PSU)
Forecast of surface water salinity
Columbia river
Estuary
River
Small et al, 1990
“CMOP: Transforming Ocean Exploration”
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Water sampling: microbiology team
Dan Murphy
• CTD, O2, Nitrate, Chl. Fluo., Trans.• DNA/RNA analysis • Dissolved nutrients (N, P, silicate)• Particulate organic carbon and nitrogen• Dissolved organic carbon• Dissolved organic nitrogen• Suspended particulate matter• Chlorophyll a and other pigments• Flow cytometry analysis• Cell counts (FISH, lugol…)• Bacterial production rates• Primary production rates
“CMOP: Transforming Ocean Exploration”
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We collected nearly 250 water samples during four research cruises in 2007.
These samples cover a broad range of salinity in the estuary, and expand the dataset collected by the CRETM LMER program in the 90s to the coastal ocean
CMOP and CRETM/LMER sampling
“CMOP: Transforming Ocean Exploration”
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CR4
CR7
CR15
CR25
CR30
CR40
Beaver Dock
15 PSU
0 PSU
Chlorophyll and bacterial production
“CMOP: Transforming Ocean Exploration”
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August 2007. Total RNA content analyzed in parallel with chlorophyll a in the context of location and depth.
•
0
5
10
15
20
25
15, 1
45, 2
3
12
22
12 2
2
50
12
11, 147
Total RNA
Chlorophyll a*
g/L
32 32 33 25 33 31 28 31 20 15 10 15 6 2 0 0 PSU
Mariya Smit
“CMOP: Transforming Ocean Exploration”
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CR4
CR7
CR15
CR25
CR30
CR40
Beaver Dock
15 PSU
0 PSU
Ammonium, Nitrate, and Nitrite
“CMOP: Transforming Ocean Exploration”
10Chlorophyll Nitrate
Ammonium Salinity
J. Needoba
De
pth
mD
ep
th m
De
pth
m
Distance km Distance kmW WE E
Silicate O2 Sat (%)
Plume, April 2007 ODV
“CMOP: Transforming Ocean Exploration”
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CR4
CR7
CR15
CR25
CR30
CR40Beaver Dock
15 PSU
0 PSU
Salinity, nitrate and phosphorus in the surface ‘fresh’ plume
“CMOP: Transforming Ocean Exploration”
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Phylogenetic composition of bacterial communities is highly variable across river to ocean gradients
St1St4
St 1: O PSU – 2 m
St4: 15 PSU – 10 m
CR4: 27 PSU – 2 m
CR4: 32 PSU – 24 m
CR40: 32 PSU – 2 m
CR40: 34 PSU – 850 m
unpubl. data from D. Murphy , P. Zuber & H. Simon (OHSU)
Single-stranded conformation
polymorphism (SSCP)
1 peak = 1 phylotype
Columbia river estuary
River
Estuary
Plume
Ocean
CR7CR40
“CMOP: Transforming Ocean Exploration”
13Multidimensional scaling diagram showing variability in
bacterioplankton assemblages based on PCR-DGGE of 16S rRNA genes.
Circled clusters are statistically significant (ANOSIM, p<0.001 for all comparisons)
Example of Denaturing Gradient Gel Electrophoresis
1 band = 1 phylotype
unpubl. data from C. Fortunado & B. Crump
(UMCES)
from Herfort et al 2007
Bacterial Community Fingerprinting
“CMOP: Transforming Ocean Exploration”
14Workflow at CMOP
Cloning/18S, 16S, cDNA
Sequencingplates
InspectionFASTA files
OHSUWashington University GSC
BLAST
FASTA filesPNNL (L. McCue, C. Oehman)
Post processing
Hit tables
Cleaninge.g., trim, sanitize
Link
Shared Knowledge
Analyze
synopsis
Cloud B. Howe, D. Maier
Hit tables + metadata
Biodemo
“CMOP: Transforming Ocean Exploration”
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DNA sequencing of 16S rDNA reveals long term consistency in river-to-ocean bacterial diversity
St4
St1
St4: 15 PSU - 10 m
CR4: 27 PSU - 2 mSt1: 0 PSU - 2 m
Alpha Proteobacteria Beta Proteobacteria
Delta Proteobacteria Gamma Proteobacteria
Actinobacterium Cyanobacteria
Bacteroidetes Verrucomicrobiales
Planctomyces Gram +
Columbia river estuary
CR4
CFB
Cyano
CFB
CFB
Actino
(CFB)
“CMOP: Transforming Ocean Exploration”
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St4
St1
St4: 15 PSU - 10 m depthData from 154 clonesCR4: 28 PSU - 2 m depth
Data from 156 clones
St1: 0 PSU - 2 m depthData from 164 clones
F u n g i
P r o tist
M et a z o a
M et a p h y t a
DNA sequencing of 18S rDNA shows heterotrophs dominate estuary, autotrophs dominate plume
Phytoplankton
“CMOP: Transforming Ocean Exploration”
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St4
St1
St4: 15 PSU - 10 m depthData from 154 clonesCR4: 28 PSU - 2 m depth
Data from 156 clones
St1: 0 PSU - 2 m depthData from 164 clones
F u n g i
P r o tist
M et a z o a
M et a p h y t a
DNA sequencing of 18S rDNA shows heterotrophs dominate estuary, autotrophs dominate plume
Phytoplankton
“CMOP: Transforming Ocean Exploration”
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River-to-ocean seasonal differences in eukaryotic microbial assemblages from 18S rDNA clone
libraries
unpubl. data from P. Kahn, L. Herfort, R. Letellier & P. Zuber
Eukaryotic Microbial Assemblages: April and August, 2007
“CMOP: Transforming Ocean Exploration”
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unpubl. data from P. Kahn, L. Herfort, P. Zuber
Diatoma tunis11%
Thalassiosira aestivalis
24%
Thalassiosira 9%
Detonula Pumila
8%
Asterionella japonica
19%
Rimostrombidium lacustris
(alveolate) 8%
Katablepharis30%
Calanus pacificus
10%
Telonema antarcticum
11%
% of the total # of clones
Eukaryotic microbial diversity
Myrionecta rubra 16%
Myrionecta rubra 11%
“CMOP: Transforming Ocean Exploration”
20EventEvent: Bloom of Myrionecta rubra
Columbia river estuarymouth
Myrionecta rubra=Mesodinium rubrum
river
Red water
KaryokleptyKaryokleptyIngest chloroplast &
nucleus from Cryptophyte algae
“CMOP: Transforming Ocean Exploration”
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Bloom of Myrionecta rubra: dynamic
Patchiness
Cast 54: 0m (left), 1m (right)Cast 54: 0m & 1m
“CMOP: Transforming Ocean Exploration”
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Bloom of M. rubra: impact on microbial diversity
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18S rDNA
16S rDNA
16S rDNA
unpubl. data from L. Herfort, M. Selby & P. Zuber
controlNear Max FluorMax. Fluor
Bacteria
Phytoplankton
Cyanobacteria
Others
“CMOP: Transforming Ocean Exploration”
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Microbial activity: cDNA library of microbial mRNA
cDNA clone library
Gene Expression
“CMOP: Transforming Ocean Exploration”
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Plume is an area of high primary productivity, bacterial productivity
Productivity of the plume fueled by nutrients delivered through tidal events
Estuary is an area of heterotrophic microbial activity and unique primary production mediated by ciliates that appropriate photosynthetic machinery (Katablepharids, Myrionecta)
Myrionecta may serve as an indicator of estuary ecosystem state as its growth correlates with low river flow and elevated temperature
Conclusions, Questions
“CMOP: Transforming Ocean Exploration”
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Microbiology Component of CMOP
Byron Crump, CMOP Investigator, UMLES Chief Scientist-Wecoma Aug 2007 Caroline Fortunato, Graduate Student, UMLES
Population analyses, DGGE 16S
Holly Simon, CMOP InvestigatorMariya Smit, Senior Research AssociateDan Murphy, Graduate Student
Prokaryotic 16S analysisCrenarchaea studiesGene expression, MicroarrayBioinformatics
Lydie Herfort, Postdoctoral Fellow, Chief Scientist-Barnes Aug 2007Wecoma Nov 2007 April 2008
Peter Kahn, Undergraduate (Willamette U.)Mikaela Selby, Technician
Prokaryotic 16SEukaryotic 18ScDNA analysis of
environmental mRNAArchaea Studies
Brad Tebo, CMOP Investigator, Head EBSSuzanna Braür, Postdoctoral FellowKira Kranzler, Undergraduate, Evergreen State University Mn oxidation, reduction 16S analysis June 2007 ETM
Joseph Needoba, Sensors, Nutrient analysisTawnya Peterson, Chief Scientist- Wecoma
May June 2008Phytoplankton,
Zooplankton
Joe Jennings, OSUNutrient analysis
Ricardo Letelier, OSUChlorophyll
UMCES Analytical Labs, Maryland
“CMOP: Transforming Ocean Exploration”
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EukA EukB
97% 97%
A B
A B=BLAST result ?
Environmental 18S clone sequencing
~ 700 b
“CMOP: Transforming Ocean Exploration”
27cDNA Nov 2006 Freshwater (0 psu)
1h08 159aa AMP-dependent synthetase and ligase [Polynucleobacter sp. QLW-P1DMWA-1]1h07 156aa hypothetical protein STIAU_5972 [Stigmatella aurantiaca DW4/3-1] 1h06 130aa nudix hydrolase domain, putative MutT/nudix family protein [Frankia alni ACN14a]1h05 172aa unknown1h04 236aa unknown1h03 223aa Threonine aldolase [Flavobacterium johnsoniae UW101]1h02 139aa AMP-dependent synthetase and ligase, long chain fatty acid [Polynucleobacter sp. QLW-P1DMWA-11g12 321aa unknown1g11 172aa unknown1g10 139aa AMP-dependent synthetase and ligase [Polynucleobacter sp. QLW-P1DMWA-1]1g09 187aa transposon protein, putative, CACTA, En/Spm sub-class1g08 207aa unknown1g07 139aa AMP-dependent synthetase and ligase [Polynucleobacter sp. QLW-P1DMWA-1]1g06 160aa transposase for insertion sequence element ISRM3 [Bacteroides thetaiotaomicron VPI-5482]
1d06 213aa transpeptidase Peptidoglycan glycosyltransferase [Thermosinus carboxydivorans1d07 250aa predicted protein [Nematostella vectensis]1d08 137aa putative helicase (mRNA degradation) [marine actinobacterium PHSC20C1]1d09 163aa unknown1d10 167aa NADH-quinone oxidoreductaseF420H2:quinone oxidoreductase chain L [Halobacterium sp. NRC-1]1d11 187aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus1e02 150aa Threonine aldolase [Flavobacterium johnsoniae UW101]1e03 ABC transporter, permease component (hydroxymethylpyrimindine) [Bacillus sp. NRRL B-14911]1e04 279aa probable extra-cytoplasmic solute receptor [Ralstonia eutropha1e07 133aa unkown1h10 168aa alanine racemase domain protein [Anaeromyxobacter sp. K]1h11 160aa major facilitator superfamily MFS_1 [Thermotoga lettingae TMO]1h09 173aa TauC ABC transporter, permease component [Bacillus sp. NRRL B-14911]
“CMOP: Transforming Ocean Exploration”
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B26 217aa unknownB25 113aa hypothetical protein Bm1_19105 [Brugia malayi]B24 241aa unknownB23 155aa unknownB21 103aa unknownB16 197aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [AlgoriphagusB15 105aa unkownB14 140aa predicted protein [Physcomitrella patens subsp. patens]B13 92aa unknownA24 175aa ABC transporter, permease component [Bacillus sp. NRRL B-14911A23 224aa putative transcriptional regulator [Shewanella sediminis HAW-EB3] AraC-like (sugar-binding domain)A22 102aa unknownA21 133aa hypothetical protein MGG_04504 [Magnaporthe grisea 70-15]A16 117aa unknownA14 139aa AMP-dependent synthetase and long-chain fatty acid ligase [Polynucleobacter A12 182aa NAD-dependent epimerase/dehydratase (cinamoyl-CoA reductase) [Herpetosiphon aurantiacus1A02 135aa unknown1a03 236aa, 122aa unknown1a04 188aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus1a05 205aa unknown1a07 89aa PREDICTED: similar to MGC53049 protein [Strongylocentrotus purpuratus]
cDNA Nov 2006 Freshwater (0 psu)
1a08 209aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus1a09 160aa unknown1a10 189aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus1a11 243aa extracellular solute-binding protein, family 3 TauA (nitrate/sulfonate/bicarbonate transport system )[Rhodoferax ferrireducens1b01 59 nitrous oxide reductase [uncultured alpha proteobacterium]1b02 121aa unknown1b03 80aa unknown1b04 103aa unknown1b05 145aa unknown1b06 184aa Rh-like protein/ammonium transporter (Amt domain) [Rhodoferax ferrireducens1b08 274aa O-antigen biosynthesis protein RbfC [Riftia pachyptila endosymbiont]1b10 234aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus1b11 265aa putative DNA polymerase [Pseudomonas phage 73]1b12 163aa unknown1c01 257aa unknown1c03 157aa 2-polyprenylphenol hydroxylase and related flavodoxin oxidoreductases [Burkholderia dolosa AUO158]1c04 121aa unkown1c05 193aa unknown1c06 241aa unknown1c07 182aa unknown1c08 181aa unknown1c12 269aa hypothetical protein GDI0505 [Gluconacetobacter diazotrophicus1d04 209aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus sp. PR1]
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