1. 1 Coastal margin science: Microbiology Microbial Diversity and Microbial-mediated Activities Peter Zuber

2. “CMOP: Transforming Ocean Exploration” 2 Coastal margin science: Microbiology Microorganisms Ecosystem & human health nationalgeographic.com Human activity & climate Physical circulation and transport Chemical stressors 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

3. “CMOP: Transforming Ocean Exploration” 3 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 properties NOAA web site Sea surface temperature Image from PISCO (OSU) Fish mortality in July 2002 Grantham et al. 2004 Nature CORIE forecast Estuarine Turbidity Maximum Enhanced microbial productivity

4. “CMOP: Transforming Ocean Exploration” 4 River, estuary, plume & ocean Estuary & plume Dynamic 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 particulate organic carbon 146,000 Mt/y phytoplankton 61,000 Mt/y Primary production 17,000 Mt/y ? Salinity (PSU) Forecast of surface water salinity Columbia river Estuary River Small et al, 1990

5. “CMOP: Transforming Ocean Exploration” 5 Water sampling: microbiology team Dan Murphy CTD, O 2, 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

6. “CMOP: Transforming Ocean Exploration” 6 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

7. “CMOP: Transforming Ocean Exploration” 7 CR4 CR7 CR15 CR25 CR30 CR40 Beaver Dock 15 PSU 0 PSU Chlorophyll and bacterial production

8. “CMOP: Transforming Ocean Exploration” 8 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 2 2 2 2 50 12 11, 1 47 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

9. “CMOP: Transforming Ocean Exploration” 9 CR4 CR7 CR15 CR25 CR30 CR40 Beaver Dock 15 PSU 0 PSU Ammonium, Nitrate, and Nitrite

10. “CMOP: Transforming Ocean Exploration” 10 ChlorophyllNitrate AmmoniumSalinity J. Needoba Depth m Distance km W W E E Silicate O 2 Sat (%) Plume, April 2007 ODV

11. “CMOP: Transforming Ocean Exploration” 11 CR4 CR7 CR15 CR25 CR30 CR40 Beaver Dock 15 PSU 0 PSU Salinity, nitrate and phosphorus in the surface ‘fresh’ plume

12. “CMOP: Transforming Ocean Exploration” 12 Phylogenetic composition of bacterial communities is highly variable across river to ocean gradients St1 St4 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 CR7 CR40

13. “CMOP: Transforming Ocean Exploration” 13 Multidimensional 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

14. “CMOP: Transforming Ocean Exploration” 14 Workflow at CMOP Cloning/ 18S, 16S, cDNA Sequencing plates Inspection FASTA files OHSU Washington University GSC BLAST FASTA files PNNL (L. McCue, C. Oehman) Post processing Hit tables Cleaning e.g., trim, sanitize Link Shared Knowledge Analyze synopsis Cloud B. Howe, D. Maier Hit tables + metadata Biodemo

15. “CMOP: Transforming Ocean Exploration” 15 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 m St1: 0 PSU - 2 m Columbia river estuary CR4       CFB  Cyano CFB Actino (CFB)

16. “CMOP: Transforming Ocean Exploration” 16 St4 St1 St4: 15 PSU - 10 m depth Data from 154 clones CR4: 28 PSU - 2 m depth Data from 156 clones St1: 0 PSU - 2 m depth Data from 164 clones Fungi Protist Metazoa Metaphyta DNA sequencing of 18S rDNA shows heterotrophs dominate estuary, autotrophs dominate plume Phytoplankton

17. “CMOP: Transforming Ocean Exploration” 17 St4 St1 St4: 15 PSU - 10 m depth Data from 154 clones CR4: 28 PSU - 2 m depth Data from 156 clones St1: 0 PSU - 2 m depth Data from 164 clones Fungi Protist Metazoa Metaphyta DNA sequencing of 18S rDNA shows heterotrophs dominate estuary, autotrophs dominate plume Phytoplankton

18. “CMOP: Transforming Ocean Exploration” 18 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

19. “CMOP: Transforming Ocean Exploration” 19 unpubl. data from P. Kahn, L. Herfort, P. Zuber Diatoma tunis 11% Thalassiosira aestivalis 24% Thalassiosira 9% Detonula Pumila 8% Asterionella japonica 19% Rimostrombidium lacustris (alveolate) 8% Katablepharis 30% Calanus pacificus 10% Telonema antarcticum 11% % of the total # of clones Eukaryotic microbial diversity Myrionecta rubra 16% Myrionecta rubra 11%

20. “CMOP: Transforming Ocean Exploration” 20 Event Event : Bloom of Myrionecta rubra Columbia river estuary mouth Myrionecta rubra=Mesodinium rubrum river Red water Karyoklepty Ingest chloroplast & nucleus from Cryptophyte algae

21. “CMOP: Transforming Ocean Exploration” 21 Bloom of Myrionecta rubra: dynamic Patchiness Cast 54: 0m (left), 1m (right) Cast 54: 0m & 1m

22. “CMOP: Transforming Ocean Exploration” 22 Bloom of M. rubra: impact on microbial diversity 22 18S rDNA 16S rDNA unpubl. data from L. Herfort, M. Selby & P. Zuber controlNear Max Fluor Max. Fluor Bacteria Phytoplankton Cyanobacteria Others

23. “CMOP: Transforming Ocean Exploration” 23 Microbial activity: cDNA library of microbial mRNA cDNA clone library Gene Expression

24. “CMOP: Transforming Ocean Exploration” 24 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

25. “CMOP: Transforming Ocean Exploration” 25 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 Investigator Mariya Smit, Senior Research Associate Dan Murphy, Graduate Student Prokaryotic 16S analysis Crenarchaea studies Gene expression, Microarray Bioinformatics Lydie Herfort, Postdoctoral Fellow, Chief Scientist-Barnes Aug 2007 Wecoma Nov 2007 April 2008 Peter Kahn, Undergraduate (Willamette U.) Mikaela Selby, Technician Prokaryotic 16S Eukaryotic 18S cDNA analysis of environmental mRNA Archaea Studies Brad Tebo, CMOP Investigator, Head EBS Suzanna Braür, Postdoctoral Fellow Kira Kranzler, Undergraduate, Evergreen State University Mn oxidation, reduction 16S analysis June 2007 ETM Joseph Needoba, Sensors, Nutrient analysis Tawnya Peterson, Chief Scientist- Wecoma May June 2008 Phytoplankton, Zooplankton Joe Jennings, OSU Nutrient analysis Ricardo Letelier, OSU Chlorophyll UMCES Analytical Labs, Maryland

26. “CMOP: Transforming Ocean Exploration” 26 EukAEukB 97% A B AB= BLAST result ? Environmental 18S clone sequencing ~ 700 b

27. “CMOP: Transforming Ocean Exploration” 27 cDNA 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 unknown 1h04 236aa unknown 1h03 223aa Threonine aldolase [Flavobacterium johnsoniae UW101] 1h02 139aa AMP-dependent synthetase and ligase, long chain fatty acid [Polynucleobacter sp. QLW-P1DMWA-1 1g12 321aa unknown 1g11 172aa unknown 1g10 139aa AMP-dependent synthetase and ligase [Polynucleobacter sp. QLW-P1DMWA-1] 1g09 187aa transposon protein, putative, CACTA, En/Spm sub-class 1g08 207aa unknown 1g07 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 carboxydivorans 1d07 250aa predicted protein [Nematostella vectensis] 1d08 137aa putative helicase (mRNA degradation) [marine actinobacterium PHSC20C1] 1d09 163aa unknown 1d10 167aa NADH-quinone oxidoreductaseF420H2:quinone oxidoreductase chain L [Halobacterium sp. NRC-1] 1d11 187aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus 1e02 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 eutropha 1e07 133aa unkown 1h10 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]

28. “CMOP: Transforming Ocean Exploration” 28 B26 217aa unknown B25 113aa hypothetical protein Bm1_19105 [Brugia malayi] B24 241aa unknown B23 155aa unknown B21 103aa unknown B16 197aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus B15 105aa unkown B14 140aa predicted protein [Physcomitrella patens subsp. patens] B13 92aa unknown A24 175aa ABC transporter, permease component [Bacillus sp. NRRL B-14911 A23 224aa putative transcriptional regulator [Shewanella sediminis HAW-EB3] AraC-like (sugar-binding domain) A22 102aa unknown A21 133aa hypothetical protein MGG_04504 [Magnaporthe grisea 70-15] A16 117aa unknown A14 139aa AMP-dependent synthetase and long-chain fatty acid ligase [Polynucleobacter A12 182aa NAD-dependent epimerase/dehydratase (cinamoyl-CoA reductase) [Herpetosiphon aurantiacus 1A02 135aa unknown 1a03 236aa, 122aa unknown 1a04 188aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus 1a05 205aa unknown 1a07 89aa PREDICTED: similar to MGC53049 protein [Strongylocentrotus purpuratus] cDNA Nov 2006 Freshwater (0 psu) 1a08 209aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus 1a09 160aa unknown 1a10 189aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus 1a11 243aa extracellular solute-binding protein, family 3 TauA (nitrate/sulfonate/bicarbonate transport system )[Rhodoferax ferrireducens 1b01 59 nitrous oxide reductase [uncultured alpha proteobacterium] 1b02 121aa unknown 1b03 80aa unknown 1b04 103aa unknown 1b05 145aa unknown 1b06 184aa Rh-like protein/ammonium transporter (Amt domain) [Rhodoferax ferrireducens 1b08 274aa O-antigen biosynthesis protein RbfC [Riftia pachyptila endosymbiont] 1b10 234aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus 1b11 265aa putative DNA polymerase [Pseudomonas phage 73] 1b12 163aa unknown 1c01 257aa unknown 1c03 157aa 2-polyprenylphenol hydroxylase and related flavodoxin oxidoreductases [Burkholderia dolosa AUO158] 1c04 121aa unkown 1c05 193aa unknown 1c06 241aa unknown 1c07 182aa unknown 1c08 181aa unknown 1c12 269aa hypothetical protein GDI0505 [Gluconacetobacter diazotrophicus 1d04 209aa acetyl-CoA carboxylase, carboxyl transferase subunit beta [Algoriphagus sp. PR1]