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Sarah N. Brown Dr. Stephen Giovannoni Dr. Jang-Cheon Cho Department of Microbiology HHMI 2011

Polyphasic taxonomy of marine bacteria from the SAR11 group Ia: Pelagibacter ubiquis (strain HTCC1062) & Pelagibacter bermudensis (strain HTCC7211). Sarah N. Brown Dr. Stephen Giovannoni Dr. Jang-Cheon Cho Department of Microbiology HHMI 2011.

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Sarah N. Brown Dr. Stephen Giovannoni Dr. Jang-Cheon Cho Department of Microbiology HHMI 2011

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  1. Polyphasic taxonomy of marine bacteria from the SAR11 group Ia: Pelagibacter ubiquis (strain HTCC1062) & Pelagibacter bermudensis (strain HTCC7211) Sarah N. Brown Dr. Stephen Giovannoni Dr. Jang-Cheon Cho Department of Microbiology HHMI 2011 3-D structures of Pelagibacter ubique (2006) (Pelagibacter means "bacterium of the sea")

  2. SAR11 • The most abundant marine bacteria (~30% in euphotic) • Proteorhodopsin –containing (photoheterotroph) • Importance in biogeochemical cycles • Global climate & weather regulation (DMSP) FIGURE 1. Distribution of the SAR11 clade in the world's oceans.

  3. Background • In 2002, SAR11 cells were first isolated in seawater-based medium (no colonies produced). • Growth of Pelagibacter in artificial seawater medium (ASW)is a recent advancement. • Non-colony forming property and oligotrophy made it difficult for taxonomy • Purpose: To characterize & provide official nomenclature for SAR11 (strains 1062 & 7211) • SAR11: Candidatus Pelagibacter Fig. 4. Cultures of oligotrophic marine bacteria growing in carboys of autoclaved seawater.

  4. Polyphasic Taxonomy GENOTYPIC INFORMATION • Incorporates multiple methods for identification & description of new species • Species: the basic unit of bacterial taxonomy • SAR11 clade • Class: Alphaproteobacteria PHENOTYPIC INFORMATION

  5. Genotypic comparison: 16S rRNA • >98.7% 16S rRNA gene sequence similarity between 1062 & 7211 • Consequently, we can’t tell whether these strains are the same species. • Therefore, genomic comparisons should be performed. (Dr. Jang Cheon-Cho, 2011)

  6. ANI: A method for bacterial species demarcation • >95-96% average nucleotide identity (ANIb/ANIm) indicates ‘true (same) species’ Strains show <95-96% average nucleotide identity (ANIb/m) Different species <95-96% ANIm/b Same species Therefore, HTCC1062 & HTCC7211 represent separate genomic species Fig., m= MUMmer computer algorithm; b= BLAST algorithm (Dr. Jang Cheon-Cho, 2011)

  7. Conclusion of genotypic analysis: 2 species HTCC1062 HTCC1002 HTCC7211 HIMB5 P. ubiquis 0.001 Group Ia P. bermudensis • 1. HTCC1062→Pelagibacter ubiquis gen. nov., sp. nov. • 2. HTCC7211→ Pelagibacter bermudensis sp. nov. (Dr. Jang Cheon-Cho, 2011)

  8. Phenotypic comparisons: growth conditions • Prepare artificial seawater medium (ASW) • Salinity: w/out NaCl & w/10% NaCl • pH: adjust w/0.1M NaOH & 0.1M HCl • Add nutrients & inoculum • Dispense into 156 flasks (triplicates of each growth condition) • Incubate • Temp (°C): 4, 8, 12, 16, 20, 23, 25, & 30 • pH & salinity: 16ºC • Screen for growth

  9. SPECIFIC GROWTH RATE (µ) µmax optimum maximum optimum minimum µ = the # of divisions per cell per unit time. It depends upon growth conditions. maximum

  10. optimum µmax SPECIFIC GROWTH RATE (µ) maximum minimum µmax µmax optimum optimum maximum maximum minimum minimum

  11. SPECIFIC GROWTH RATE (µ) µmax optimum minimum maximum µmax optimum minimum maximum

  12. Final thoughts Phenotype Summary Conclusions • Growth data suggest that these strains are separate species. • Genotypic & phenotypic data show that these are 2 distinct species. • Therefore, we propose the following nomenclature : • Strain HTCC1062→ Pelagibacter ubiquis gen. nov., sp. nov. • Strain HTCC7211→ Pelagibacter bermudensis sp. nov.

  13. Acknowledgments • The Gordon & Betty Moore Foundation • Dr. Stephen Giovannoni • Dr. Jang Cheon-Cho • Paul Carini • Kevin Vergin • Giovannoni Lab • HHMI & Dr. Kevin Ahern

  14. References • Konstantinidis, K., and J. M. Tiedje. 2005. Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. USA 102:2567-2572. • Konstantinidis, K., and J. M. Tiedje. 2005. Towards a genome-based taxonomy for prokaryotes. J. Bacteriol. 187:6258-6264. • Morris, R.M., Rappé, M.S., Connon,S.A., Vergin, K.L., Siebold, W.A., Carlson, C.A., and Giovannoni, S.J. (December 2002). SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420: 806-810. doi:10.1038/nature01240. • Nicastro, D., Schwartz, C., Pierson, J., Cho, J.-C.C., Giovannoni, S. J., and McIntosh, J. R. (2006). Three-dimensional structure of the tiny bacterium Pelagibacter ubique studied by cryo-electron tomography. Microsc. Microanal. 12(sup2):180-181. • Richter, M., Rosselló-Móra, R. (October 2009). Shifting the genomic gold standard for the prokaryotic species definition. Biological Sciences - Microbiology: PNAS 106 (45): 19126-19131; doi:10.1073/pnas.0906412106 .

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