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
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")

2. 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.

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
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.

4. 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

5. 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)

6. 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)

7. 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)

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
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.

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

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

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

18. 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

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

20. References
Konstantinidis, K., and J. M. Tiedje Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. USA 102:
Konstantinidis, K., and J. M. Tiedje Towards a genome-based taxonomy for prokaryotes. J. Bacteriol. 187:
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: doi: /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):
Richter, M., Rosselló-Móra, R. (October 2009). Shifting the genomic gold standard for the prokaryotic species definition. Biological Sciences - Microbiology: PNAS 106 (45): ; doi: /pnas