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

5.  >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. 16S rRNA gene sequence similarity HTCC7211 HTCC106298.9% (Dr. Jang Cheon-Cho, 2011)

6.  >95-96% average nucleotide identity (ANI b /ANI m ) indicates ‘true (same) species’ Fig., m= MUMmer computer algorithm; b= BLAST algorithm ANI b HTCC7211 HTCC106276.73 ANI m HTCC7211 HTCC106282.61 Strains show <95-96% average nucleotide identity (ANI b/m ) Therefore, HTCC1062 & HTCC7211 represent separate genomic species (Dr. Jang Cheon-Cho, 2011) Different species <95-96% ANIm/b Same species

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

8.  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

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

14. µ max minimum optimum maximum µ max optimum minimum maximum optimum maximum minimum µ max

17. minimum optimum maximum SPECIFIC GROWTH RATE (µ) minimum µ max optimum maximum

18. 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. Optimum Growth Conditions Oregon Coast Strain HTCC1062 Sargasso Sea Strain HTCC7211 Temperature16ºC23ºC Salinity1.5% NaCl2% NaCl pH6.58

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

20.  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.