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 Alternative sources of energy / clean the environment Isolated by Dr. Uli Stingl from the Sargasso Sea Isolated by Dr. Uli Stingl from the Sargasso Sea Maximum density in culture is between 10 5 and10 6 cells/mL Maximum density in culture is between 10 5 and10 6 cells/mL

Background No additional growth when inoculated into seawater containing 90 different carbon compounds No additional growth when inoculated into seawater containing 90 different carbon compounds Oregon vs. Bermuda coastal waters Oregon vs. Bermuda coastal waters Analysis of 16s rRNA – related to thiotrophic symbionts Analysis of 16s rRNA – related to thiotrophic symbionts

Questions to Explore Which compound(s) does HTCC5015 use as a carbon source? Which compound(s) does HTCC5015 use as a carbon source? Are HTCC5015’s energy needs similar to that of thiotrophic symbionts? 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. Determine HTCC5015’s nutrient requirements therefore achieving a higher maximum cell density in culture.

Growth Experiment 1 Test three separate compounds + control (LNHM) 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

Growth Experiment 2 Test two compounds separately + control (sea water) Test two compounds separately + control (sea water) – LNHM – Potassium Nitrate Base media containing filtered sea water and fortified with vitamins, iron, phosphorus, and nitrogen – in the form of NH 3 Experimental source of nitrogen in replacement of ammonia

Growth Experiment 2 Control

Growth Experiment 2 Results

Growth Experiment 3 Test five compounds separately + control (LNHM) Test five compounds separately + control (LNHM) – Glucose – Succinate – Pyruvate – Sodium Acetate – D-ribose Possible carbon sources

Growth Experiment 3 Control

Growth Experiment 3 Results

Growth Experiment 4 Test five compounds separately+ control (LNHM) 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

Continuation Re-test sulfur compounds from experiment 4 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 Analyze HTCC5015 genome to determine incomplete metabolic pathways – Formulate experiments with compounds unable to be made by HTCC5015

Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute Kevin Ahern Kevin Ahern Stephen Giovannoni Stephen Giovannoni Kevin Vergin Kevin Vergin Jim Tripp Jim Tripp Tom Richardson Tom Richardson Joshua Kitner Joshua Kitner Uli Stingl Uli Stingl