Sarah N. Brown
Dr. Stephen Giovannoni
Dr. Jang-Cheon Cho
Department of Microbiology
HHMI 2011
3-D structures of Pelagibacter ubique (2006)
(Pelagibacter means "bacterium of the sea")

The most abundant marine
bacteria (~30% in euphotic)

Importance in biogeochemical
cycles

Proteorhodopsin –containing
(photoheterotroph)

Global climate & weather
regulation (DMSP)
FIGURE 1. Distribution of the SAR11 clade in the world's oceans.

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.

Incorporates multiple methods for
identification & description of
new species

Species: the basic unit of
bacterial taxonomy

SAR11 clade
•  Class: Alphaproteobacteria
GENOTYPIC INFORMATION
PHENOTYPIC INFORMATION
16S rRNA gene sequence similarity

>98.7% 16S rRNA gene sequence
similarity between 1062 & 7211

Consequently, we can’t tell whether
these strains are the same species.
HTCC7211
HTCC1062
98.9%
Therefore, genomic comparisons
should be performed.
(Dr. Jang Cheon-Cho, 2011)

>95-96% average nucleotide identity (ANIb
/ANIm) indicates ‘true (same) species’
Different species
<95-96% ANIm/b
Same species
Fig., m= MUMmer computer algorithm; b= BLAST algorithm
(Dr. Jang Cheon-Cho, 2011)
ANIb
HTCC1062
HTCC7211
76.73
ANIm
HTCC1062
HTCC7211
82.61
Strains show <95-96% average
nucleotide identity (ANIb/m)
Therefore, HTCC1062 & H
TCC7211 represent separat
e genomic species
HTCC1062
P. ubiquis
HTCC1002
HTCC7211 P. bermudensis
HIMB5
0.001
1. HTCC1062→ Pelagibacter ubiquis gen. nov., sp. nov.
2. HTCC7211→ Pelagibacter bermudensis sp. nov.
(Dr. Jang Cheon-Cho, 2011)
Group Ia

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
The Effects of Temperature on the Average Growth
Rate of HTCC1062
LOG . OF NO. OF BACTERIA PER ML
1.00E+08 8C
23C
8C
12C
16C
20C
23C
25C
30C
12C
1.00E+07 20C
16C
25C
1.00E+06 1.00E+05 30C
1.00E+04 0 5 10 15 20 25 TIME (days)
30 35 40 45 The Effects of Temperature on the Average Growth
Rate of HTCC7211
1.00E+08
16C
25C
Cells/mL (Log scale)
20C
23C
1.00E+07
23C
4C
8C
12C
16C
16C
12C
1.00E+06
8C
20C
23C
4C
1.00E+05
25C
30C
30C
1.00E+04
0 5 10 15 20 Time (days)
25 30 35 0.6
µmax
optimum
16
0.4
20
23
minimum
0.2
12
25
8
(µ)
0
6
-0.2
10
14
18
22
maximum 30
26
30
Temperature vs. specific growth rate of
HTCC7211
µmax
optimum
0.3
TEMPERATURE (ºC)
23
µ = the # of divisions per cell
per unit time. It depends
upon growth conditions.
SPECIFIC GROWTH RATE (µ)
SPECIFIC GROWTH RATE (µ)
SPECIFIC
GROWTH
RATE
Temperature vs. specific growth rate
of HTCC1062
16
20
25
0.2
0.1 minimum
8
12
4
1E-16
1
-0.1
3
5
TEMPERATURE (ºC)
7
9
maximum 30
The Effects of NaCl Concentration on the Average Growth Rate of
HTCC1062
1.00E+08
2.0% NaCl
1.5% NaCl
Cells/mL (Log scale)
2.5% NaCl
1.00E+07
3.0% NaCl
2.8% NaCl
1% NaCl
1.5% NaCl
2.0% NaCl
2.5% NaCl
2.8% NaCl
3.0% NaCl
3.5% NaCl
4.0% NaCl
4.5% NaCl
1% NaCl
4.0% NaCl
4.5% NaCl
1.00E+06
1.00E+05
0 5 10 15 20 Time (days)
25 30 35 The Effects of NaCl Concentration on the Average
Growth Rate of HTCC7211
Cells/mL (Log scale) 1.00E+08 0% NaCl
1.5% NaCl 2.5% NaCl 1.00E+07 3.0% NaCl 3.5% NaCl 0.5% NaCl
1.0% NaCl
2% NaCl 1.5% NaCl
1.00E+06 4% NaCl 2% NaCl
1.00E+05 4.5% NaCl 1.0% NaCl 0% NaCl 1.00E+04 2.5% NaCl
3.0% NaCl
0.5% NaCl 3.5% NaCl
4% NaCl
4.5% NaCl
1.00E+03 0 5 10 15 20 Time (days) 25 30 35 % NaCl vs. specific growth rate of HTCC1062 µmax
optimum
1.5 2 0.6 2.5 3 2.8 3.5 0.5 4 0.4 maximum 4.5 minimum
1 0.3 1 1.5 2 2.5 3 3.5 4 4.5 5 NaCl (%) % NaCl vs. specific growth rate of
HTCC7211
0.4
µmax
µmax
optimum
optimum
0.3
SPECIFIC GROWTH RATE (µ)
SPECIFIC GROWTH RATE (µ) 0.7 2
2.5
3
1.5
0.2
3.5
0.1
4
0
0
1
0
0.5
-0.1minimum
minimum
-0.2
2
1
% NaCl
3
maximum 4.5
4
5
The Effects of pH on the Average Growth Rate of
HTCC1062
1.00E+09
Cells/mL (log scale)
1.00E+08
pH 5.5
pH 7.0
pH 5
pH 5.5
pH 6
pH 6.5
pH 7.0
pH 7.5
pH 7.8
pH 8.0
pH 8.5
pH 9.0
pH 6.5
pH 6
pH 7.5
1.00E+07
pH 7.8
pH 5
pH 8.0
pH 6
1.00E+06
pH 9.0
1.00E+05
pH 8.5
1.00E+04
1.00E+03
0
5
10
15
20
Time (days)
25
30
35
The Effects of pH on the Average Growth Rate of
HTCC7211
1.00E+08
Cells/mL (log scale)
pH 8.0
1.00E+07
pH 5.5
pH 7.5
pH 7
pH 6.0
1.00E+06
pH 8.5
1.00E+05
pH 5
1.00E+04
pH 9.0
1.00E+03
0 5 10 15 20 Time (days)
25 30 35 pH 5
pH 5.5
pH 6.0
pH 7
pH 7.5
pH 8.0
pH 8.5
pH 9.0
pH effects on the specific growth rate of HTCC1062 0.6 optimum
5.5 0.4 0.3 6 6.5 7 7.5 7.8 8 minimum
5 0.2 0.1 0 ‐0.1 5 5.5 6 6.5 7 8 8.5 9 maximum9 ‐0.2 ‐0.3 7.5 8.5 pH effects on the specific growth rate of
HTCC7211
pH 0.4
SPECIFIC GROWTH RATE (µ)
SPECIFIC GROWTH RATE (µ) µmax
0.5 µmax
optimum
8
0.3
5.5
0.2
7
6
7.5
0.1
minimum
0
1
2 5
3
4
5
6
7
8
8.5
9
10
-0.1
maximum
-0.2
-0.3
9
pH
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.

Optimum
Growth
Conditions
Oregon Coast
Strain
HTCC1062
Sargasso Sea
Strain
HTCC7211
Temperature
16ºC
23ºC
Salinity
1.5% NaCl
2% NaCl
pH
6.5
8
•  Strain HTCC7211→
Pelagibacter bermudensis sp. nov.
  The
Gordon & Betty
Moore Foundation
  Dr. Stephen Giovannoni
  Dr. Jang Cheon-Cho
  Paul Carini
  Kevin Vergin
  Giovannoni Lab
  HHMI & Dr. Kevin Ahern

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 .