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

HTCC7211

HTCC1062 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

20C

16C

12C

8C

The Effects of Temperature on the Average Growth Rate of HTCC1062

8C12C16C20C23C25C30C

TIME (days)

LO

G .

OF N

O.

OF B

AC

TER

IA P

ER

ML

0 5 10 15 20 25 30 351.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

30C

25C

23C

23C

20C

16C

16C

12C

8C

4C

The Effects of Temperature on the Average Growth Rate of HTCC7211

4C

8C

12C

16C

20C

23C

25C

30C

Time (days)

Ce

lls/m

L (

Lo

g s

ca

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)

SP

EC

IFIC

GR

OW

TH

RA

TE (

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)

SP

EC

IFIC

GR

OW

TH

RA

TE (

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% NaCl

2.8% NaCl2.5% 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)

Ce

lls/m

L (

Lo

g s

ca

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

sca

le)

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

SP

EC

IFIC

GR

OW

TH

RA

TE (

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

minimummaximum

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 7.8

pH 7.5pH 7.0

pH 6.5

pH 6

pH 6

pH 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/m

L

(log s

cale

)

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)

Ce

lls/m

L (

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

SP

EC

IFIC

GR

OW

TH

RA

TE (

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

µmaxoptimum

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% 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

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 .