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’
ANIb
HTCC1062
HTCC7211
76.73
ANIm
HTCC1062
Different species
<95-96% ANIm/b
Same species
Fig., m= MUMmer computer algorithm; b= BLAST algorithm
(Dr. Jang Cheon-Cho, 2011)
HTCC7211
82.61
Strains show <95-96% average
nucleotide identity (ANIb/m)
Therefore, HTCC1062 &
HTCC7211 represent separate
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
8C
12C
16C
16C
12C
1.00E+06
4C
20C
8C
23C
25C
4C
1.00E+05
30C
30C
1.00E+04
0
5
10
15
20
Time (days)
25
30
35
SPECIFIC
GROWTH
RATE
Temperature vs. specific growth rate
of HTCC1062
0.6
optimum
16
0.4
20
23
minimum
0.2
12
25
8
(µ)
0
6
-0.2
10
14
18
22
maximum
26
30 30
Temperature vs. specific growth rate of
HTCC7211
TEMPERATURE (ºC)
µmax
0.3
optimum 23
16
µ = the # of divisions per cell
per unit time. It depends
upon growth conditions.
SPECIFIC GROWTH RATE (µ)
SPECIFIC GROWTH RATE (µ)
µmax
20
25
0.2
8
minimum
0.1
12
4
0
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
3.0% NaCl
1.0% NaCl
0.5% NaCl
0% NaCl
2.5% NaCl
1.00E+04
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 vs. specific growth rate of
HTCC7211
NaCl (%)
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
0
0.5
-0.1minimum
minimum
-0.2
1
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 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 7.0
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
pH 5.5
Cells/mL (log scale)
pH 8.0
1.00E+07
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
µ0.5
max
optimum 6.5
7.5
0.4
7.8 8
minimum
5
0.3
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)
7
6
5.5
0.3
µmax
optimum
8
5.5
0.2
7
6
7.5
0.1
minimum
0
1
5
3
5
7
8.5
9
-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 .