Characterization of HTCC5015: A
Novel Bacterial Isolate from the
Sargasso Sea
By: Marie Johnson
Dr. Stephen Giovannoni Laboratory of
Marine Microbiology
Background
• Alternative sources of energy / clean the
environment
• Isolated by Dr. Uli Stingl from the Sargasso Sea
• Maximum density in culture is between 105 and106
cells/mL
Background
• No additional growth when inoculated into seawater
containing 90 different carbon compounds
• Oregon vs. Bermuda coastal waters
• Analysis of 16s rRNA – related to thiotrophic
symbionts
Questions to Explore
• Which compound(s) does HTCC5015 use as a carbon
source?
• Are HTCC5015’s energy needs similar to that of
thiotrophic symbionts?
Strategy
• Determine HTCC5015’s nutrient requirements
therefore achieving a higher maximum cell
density in culture.
Growth Experiment 1
• Test three separate compounds + control
(LNHM)
– DMSP
– Glycine Betaine
– Choline
Sulfur compounds possibly used in
metabolic processes
Possible carbon source
Growth Experiment 1
Control
Growth Experiment 1 Results
5015 Growth Curve (averages)
Density (Cells/mL)
4.50E+05
4.00E+05
control
3.50E+05
DMSP 10 μM
DMSP 5 μM
3.00E+05
DMSP 1 μM
2.50E+05
Glycine Betaine 10 μM
2.00E+05
Glycine Betaine 5 μM
1.50E+05
Glycine Betaine 1 μM
Choline 10 μM
1.00E+05
Choline 5 μM
5.00E+04
Choline 1 μM
0.00E+00
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (Hours) after Inoculation
Cell Density (cells/mL)
Maximum Cell Density for Experimental Compounds
5.00E+05
4.50E+05
4.00E+05
3.50E+05
3.00E+05
2.50E+05
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
Contents of Growth Medium
control
DMSP 10 μM
DMSP 5 μM
DMSP 1 μM
Glycine Betaine 10 μM
Glycine Betaine 5 μM
Glycine Betaine 1 μM
Choline 10 μM
Choline 5 μM
Choline 1 μM
Growth Experiment 2
• Test two compounds separately + control (sea
water)
– LNHM
Base media containing filtered sea water and fortified with
vitamins, iron, phosphorus, and nitrogen – in the form of NH3
– Potassium Nitrate
Experimental source of nitrogen in
replacement of ammonia
Growth Experiment 2
Control
Growth Experiment 2 Results
Density (Cells/mL)
5015 Growth Curve (averages)
4.50E+05
SW
4.00E+05
LNHM
3.50E+05
KNO3 10μM
3.00E+05
KNO3 5μM
2.50E+05
KNO3 1μM
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
0
2
4
6
8
10
12
14
16
18
20
22
Time (Hours)
Cell Density (cells/mL)
Maximum Cell Density for Experimental Compounds
5.00E+05
4.50E+05
4.00E+05
3.50E+05
3.00E+05
2.50E+05
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
SW
LNHM
KNO3 10μM
KNO3 5μM
KNO3 1μM
Contents of Growth Medium
Growth Experiment 3
• Test five compounds separately + control
(LNHM)
– Glucose
– Succinate
– Pyruvate
– Sodium Acetate
– D-ribose
Possible carbon sources
Growth Experiment 3
Control
Growth Experiment 3 Results
Density (Cells/mL)
5015 Growth Curve (averages)
5.00E+05
LNHM
4.50E+05
Glucose 1μM
4.00E+05
D-ribose 1μM
Succinate 1μM
3.50E+05
Pyruvate 1μM
3.00E+05
Acetate 1μM
2.50E+05
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
Time (Hours)
Cell Density (cells/mL)
Maximum Cell Density for Experimental Compounds
5.00E+05
4.50E+05
4.00E+05
3.50E+05
3.00E+05
2.50E+05
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
LNHM
Glucose 1μM
D-ribose 1μM
Succinate 1μM
Pyruvate 1μM
Acetate 1μM
Contents of Growth Medium
Growth Experiment 4
• Test five compounds separately+ control
(LNHM)
– DMSP
– Cystine
– Methionine
– Cysteine
– Thiosulfate
Sulfur compounds possibly used in
metabolic processes
Growth Experiment 4
Control
Growth Experiment 4 Results
Density (Cells/mL)
5015 Growth Curve (averages)
2.40E+06
2.25E+06
2.10E+06
1.95E+06
1.80E+06
1.65E+06
1.50E+06
1.35E+06
1.20E+06
1.05E+06
9.00E+05
7.50E+05
6.00E+05
4.50E+05
3.00E+05
1.50E+05
0.00E+00
control
DMSP 50nM
DMSP100nM
Methionine 50nM
Methionine 100nM
Cysteine 50nM
Cysteine 100nM
Cystine 50nM
Cystine 100nM
Thiosulfate 50nM
Thiosulfate 100nM
0
2
4
6
8
10
12
14
16
18
20
22
Time (Hours)
Cell Density (cells/mL)
Maximum Cell Density for Experimental Compounds
3.00E+06
2.70E+06
2.40E+06
2.10E+06
1.80E+06
1.50E+06
1.20E+06
9.00E+05
6.00E+05
3.00E+05
0.00E+00
Contents of Growth Medium
control
DMSP 50nM
DMSP100nM
Methionine 50nM
Methionine 100nM
Cysteine 50nM
Cysteine 100nM
Cystine 50nM
Cystine 100nM
Thiosulfate 50nM
Thiosulfate 100nM
Continuation
• Re-test sulfur compounds from experiment 4
– Find support to either accept or reject the previous results
that providing cystine may allow for an increased
maximum density in culture
• Analyze HTCC5015 genome to determine incomplete
metabolic pathways
– Formulate experiments with compounds unable to be
made by HTCC5015
Acknowledgements
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Howard Hughes Medical Institute
Kevin Ahern
Stephen Giovannoni
Kevin Vergin
Jim Tripp
Tom Richardson
Joshua Kitner
Uli Stingl