sarah n. brown dr. stephen giovannoni dr. jang-cheon cho department of microbiology hhmi 2011
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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 .