Coastal margin science: Microbiology Microbial Diversity and Microbial-mediated Activities

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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


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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


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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


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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


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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


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CR4

CR7

CR15

CR25

CR30

CR40

Beaver Dock

15 PSU

0 PSU

Chlorophyll and bacterial production


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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


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CR4

CR7

CR15

CR25

CR30

CR40

Beaver Dock

15 PSU

0 PSU

Ammonium, Nitrate, and Nitrite


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


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CR4

CR7

CR15

CR25

CR30

CR40Beaver Dock

15 PSU

0 PSU

Salinity, nitrate and phosphorus in the surface ‘fresh’ plume


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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


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


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


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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)


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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


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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


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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


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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%


20EventEvent: Bloom of Myrionecta rubra

Columbia river estuarymouth

Myrionecta rubra=Mesodinium rubrum

river

Red water

KaryokleptyKaryokleptyIngest chloroplast &

nucleus from Cryptophyte algae


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Bloom of Myrionecta rubra: dynamic

Patchiness

Cast 54: 0m (left), 1m (right)Cast 54: 0m & 1m


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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


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Microbial activity: cDNA library of microbial mRNA

cDNA clone library

Gene Expression


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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


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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


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EukA EukB

97% 97%

A B

A B=BLAST result ?

Environmental 18S clone sequencing

~ 700 b


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]


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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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Coastal margin science: Microbiology Microbial Diversity and Microbial-mediated Activities