Polyphasic taxonomy of marine bacteria from the SAR11 group Ia:

Pelagibacter ubiquis (strain HTCC1062) & Pelagibacter bermudensis (strain HTCC7211)

Sarah N. Brown

Dr. Stephen GiovannoniDr. Jang-Cheon Cho

Department of Microbiology HHMI 2011

3-D structures of Pelagibacter ubique (2006)(Pelagibacter means "bacterium of the sea")

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.

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.

Polyphasic Taxonomy Incorporates multiple

methods for identification & description of new species

Species: the basic unit of bacterial taxonomy

SAR11 clade• Class:

Alphaproteobacteria

GENOTYPIC INFORMATION

PHENOTYPIC INFORMATION

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.

16S rRNA gene sequence similarity

HTCC7211HTCC1062 98.9%

(Dr. Jang Cheon-Cho, 2011)

ANI: A method for bacterial species demarcation

>95-96% average nucleotide iden-tity (ANIb/ANIm) indicates ‘true (same) species’

Fig., m= MUMmer computer algorithm; b= BLAST algorithm

ANIb 　 HTCC7211HTCC1062 76.73

ANIm 　 HTCC7211HTCC1062 82.61

Strains show <95-96% average nucleotide identity (ANIb/m)

Therefore, HTCC1062 & HTCC7211 represent separate

genomic species

(Dr. Jang Cheon-Cho, 2011)

Different species <95-96% ANIm/b Same species

HTCC1062

HTCC1002

HTCC7211

HIMB5

0.001

Conclusion of genotypic analysis: 2 species

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

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

0 5 10 15 20 25 30 35 40 451.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

30C

25C

23C

20C16C

12C

8C

The Effects of Temperature on the Average Growth Rate of HTCC1062

8C12C16C20C23C25C30C

TIME (days)

LOG

. O

F N

O. O

F BA

CTER

IA P

ER M

L

0 5 10 15 20 25 30 351.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

30C

25C

23C23C

20C

16C

16C

12C

8C

4C

The Effects of Temperature on the Average Growth Rate of HTCC7211

4C8C12C16C20C23C25C30C

Time (days)

Cells

/mL

(Log

sca

le)

1 3 5 7 9

-0.1

-2.77555756156289E-17

0.1

0.2

0.3

Temperature vs. specific growth rate of HTCC7211

TEMPERATURE (ºC)

SPEC

IFIC

GRO

WTH

RAT

E (µ

)µmax

6 10 14 18 22 26 30

-0.2

0

0.2

0.4

0.6

Series1

Temperature vs. specific growth rate of HTCC1062

TEMPERATURE (ºC)

SPEC

IFIC

GRO

WTH

RAT

E (µ

)

minimum

µmax optimum

maximum

minimum

optimum

maximum

SPECIFIC GROWTH

RATE(µ)

µ = the # of divisions per cell per unit time. It depends upon growth conditions.

0 5 10 15 20 25 30 351.00E+05

1.00E+06

1.00E+07

1.00E+08

4.5% NaCl

4.0% NaCl

3.0% NaCl2.8% NaCl

2.5% NaCl

2.0% NaCl

1.5% NaCl

1% NaCl

The Effects of NaCl Concentration on the Av-erage Growth Rate of HTCC1062

1% NaCl1.5% NaCl2.0% NaCl2.5% NaCl2.8% NaCl3.0% NaCl3.5% NaCl4.0% NaCl4.5% NaCl

Time (days)

Cells

/mL

(Log

sca

le)

0 5 10 15 20 25 30 351.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

4.5% NaCl

4% NaCl

3.5% NaCl3.0% NaCl

2.5% NaCl

2% NaCl

1.5% NaCl

1.0% NaCl0.5% NaCl

0% NaCl

The Effects of NaCl Concentration on the Average Growth Rate of HTCC7211

0% NaCl0.5% NaCl1.0% NaCl1.5% NaCl2% NaCl2.5% NaCl3.0% NaCl3.5% NaCl4% NaCl

Time (days)

Cells

/mL (

Log

scal

e)

µmax

minimum

optimum

maximum

SPECIFIC GROWTH

RATE(µ)

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

-0.2

-0.1

0

0.1

0.2

0.3

0.4

Series1

% NaCl vs. specific growth rate of HTCC7211

% NaCl

SPEC

IFIC

GRO

WTH

RAT

E (µ

)

1 1.5 2 2.5 3 3.5 4 4.5 50.3

0.4

0.5

0.6

0.7

Series1

% NaCl vs. specific growth rate of HTCC1062

NaCl (%)

SPEC

IFIC

GRO

WTH

RAT

E (µ

) µmaxoptimum

minimum maximum

optimum

maximum

minimum

µmax

0 5 10 15 20 25 30 351.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

pH 9.0

pH 8.5

pH 8.0pH 7.8pH 7.5

pH 7.0pH 6.5

pH 6

pH 6pH 5.5

pH 5

The Effects of pH on the Average Growth Rate of HTCC1062

pH 5pH 5.5pH 6pH 6.5pH 7.0pH 7.5pH 7.8pH 8.0pH 8.5pH 9.0

Time (days)

Cells

/mL

(lo

g sc

ale)

0 5 10 15 20 25 30 351.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

pH 9.0

pH 8.5

pH 8.0

pH 7.5pH 7pH 6.0

pH 5.5

pH 5

The Effects of pH on the Average Growth Rate of HTCC7211

pH 5pH 5.5pH 6.0pH 7pH 7.5pH 8.0pH 8.5pH 9.0

Time (days)

Cells

/mL

(log

sca

le)

1 2 3 4 5 6 7 8 9 10

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

pH effects on the specific growth rate of HTCC7211

pH

SPEC

IFIC

GRO

WTH

RAT

E (µ

)

5 5.5 6 6.5 7 7.5 8 8.5 9

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

Series1

pH effects on the specific growth rate of HTCC1062

pH

SPEC

IFIC

GRO

WTH

RAT

E (µ

)

µmax

minimum

optimum

maximum

SPECIFIC GROWTH

RATE(µ)

minimum

µmax optimum

maximum

Final thoughtsPHENOTYPE 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% NaClpH 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

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 .