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 The name of the genus (Pelagibacter) stems from the Latin masculin noun pelagus ("sea") combined with the suffix –bacter (rod, bacterium), to mean "bacterium of the sea". 3-D structures of Pelagibacter ubique (2006) (Pelagibacter means "bacterium of the sea")

FIGURE 1. Distribution of the SAR11 clade in the world's oceans. 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.

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 Two marine bacterial isolates (HTCC7211 & HTCC1062) from different regions (the Pacific ocean & Sargasso sea) were studied to determine their evolutionary relationship and phylogenetic position using a polyphasic taxonomic approach, including genotypic analyses & phenotypic characterization. Fig. 4. Cultures of oligotrophic marine bacteria growing in carboys of autoclaved seawater.

Polyphasic Taxonomy Incorporates multiple methods for identification & description of new species Species: the basic unit of bacterial taxonomy SAR11 clade Class: Alphaproteobacteria GENOTYPIC INFORMATION Domain: Bacteria Phylum: Proteobacteria Class: Alphaproteobacteria Order: Rickettsiales Family: Pelagibacteraceae, fam. nov. (‘The SAR11 clade’) Genus: Pelagibacter Species: P. ubique PHENOTYPIC INFORMATION

Genotypic comparison: 16S rRNA 16S rRNA gene sequence similarity HTCC7211 HTCC1062 98.9% >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)

ANI: A method for bacterial species demarcation >95-96% average nucleotide identity (ANIb/ANIm) indicates ‘true (same) species’ ANIb HTCC7211 HTCC1062 76.73 ANIm 82.61 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)

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

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 Grow cells in a uniform ASW-based medium enriched with iron, vitamins, ammonium, and phosphate, then manipulate a single treatment condition (temperature, pH, or salinity) and screen for cell density.

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

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

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

Oregon Coast Strain HTCC1062 Sargasso Sea Strain HTCC7211 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. Optimum Growth Conditions Oregon Coast Strain HTCC1062 Sargasso Sea Strain HTCC7211 Temperature 16ºC 23ºC Salinity 1.5% NaCl 2% NaCl pH 6.5 8

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

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