Characterization of HTCC5015: A Characterization of HTCC5015: A Novel Bacterial Isolate from the Novel Bacterial Isolate from the

Sargasso SeaSargasso Sea

By: Marie JohnsonBy: Marie JohnsonDr. Stephen Giovannoni Laboratory of Dr. Stephen Giovannoni Laboratory of

Marine MicrobiologyMarine Microbiology

BackgroundBackground

• Alternative sources of energy / clean the Alternative sources of energy / clean the environment environment

• Isolated by Dr. Uli Stingl from the Sargasso SeaIsolated by Dr. Uli Stingl from the Sargasso Sea

• Maximum density in culture is between 10Maximum density in culture is between 1055 and10 and106 6

cells/mL cells/mL

BackgroundBackground

• No additional growth when inoculated into seawater No additional growth when inoculated into seawater containing 90 different carbon compoundscontaining 90 different carbon compounds

• Oregon vs. Bermuda coastal watersOregon vs. Bermuda coastal waters

• Analysis of 16s rRNA – related to thiotrophic Analysis of 16s rRNA – related to thiotrophic symbionts symbionts

Questions to ExploreQuestions to Explore• Which compound(s) does HTCC5015 use as a carbon Which compound(s) does HTCC5015 use as a carbon

source?source?

• Are HTCC5015’s energy needs similar to that of Are HTCC5015’s energy needs similar to that of thiotrophic symbionts?thiotrophic symbionts?

StrategyStrategy

• Determine HTCC5015’s nutrient requirements Determine HTCC5015’s nutrient requirements therefore achieving a higher maximum cell therefore achieving a higher maximum cell density in culture. density in culture.

Growth Experiment 1Growth Experiment 1

• Test three separate compounds + control Test three separate compounds + control (LNHM)(LNHM)

– DMSP DMSP – Glycine BetaineGlycine Betaine

– CholineCholine

Sulfur compounds possibly used in Sulfur compounds possibly used in metabolic processesmetabolic processes

Possible carbon sourcePossible carbon source

Growth Experiment 1Growth Experiment 1ControlControl

Growth Experiment 1 ResultsGrowth Experiment 1 Results

Growth Experiment 2Growth Experiment 2

• Test two compounds separately + control (sea Test two compounds separately + control (sea water)water)

– LNHM LNHM

– Potassium Nitrate Potassium Nitrate

Base media containing filtered sea water and fortified with Base media containing filtered sea water and fortified with vitamins, iron, phosphorus, and nitrogen – in the form of NHvitamins, iron, phosphorus, and nitrogen – in the form of NH33

Experimental source of nitrogen in Experimental source of nitrogen in replacement of ammoniareplacement of ammonia

Growth Experiment 2Growth Experiment 2ControlControl

Growth Experiment 2 ResultsGrowth Experiment 2 Results

Growth Experiment 3Growth Experiment 3

• Test five compounds separately + control Test five compounds separately + control (LNHM)(LNHM)

– Glucose Glucose – SuccinateSuccinate– PyruvatePyruvate– Sodium AcetateSodium Acetate– D-riboseD-ribose

Possible carbon sourcesPossible carbon sources

Growth Experiment 3Growth Experiment 3ControlControl

Growth Experiment 3 ResultsGrowth Experiment 3 Results

Growth Experiment 4Growth Experiment 4

• Test five compounds separately+ control Test five compounds separately+ control (LNHM)(LNHM)

– DMSPDMSP– CystineCystine– MethionineMethionine– CysteineCysteine– ThiosulfateThiosulfate

Sulfur compounds possibly used in Sulfur compounds possibly used in metabolic processesmetabolic processes

Growth Experiment 4Growth Experiment 4ControlControl

Growth Experiment 4 ResultsGrowth Experiment 4 Results

ContinuationContinuation

• Re-test sulfur compounds from experiment 4Re-test sulfur compounds from experiment 4– Find support to either accept or reject the previous results Find support to either accept or reject the previous results

that providing cystine may allow for an increased that providing cystine may allow for an increased maximum density in culturemaximum density in culture

• Analyze HTCC5015 genome to determine incomplete Analyze HTCC5015 genome to determine incomplete metabolic pathwaysmetabolic pathways– Formulate experiments with compounds unable to be Formulate experiments with compounds unable to be

made by HTCC5015 made by HTCC5015

AcknowledgementsAcknowledgements

• Howard Hughes Medical InstituteHoward Hughes Medical Institute• Kevin AhernKevin Ahern• Stephen Giovannoni Stephen Giovannoni • Kevin VerginKevin Vergin• Jim TrippJim Tripp• Tom RichardsonTom Richardson• Joshua KitnerJoshua Kitner• Uli StinglUli Stingl