Honors College Thesis
 

A genomic and proteomic characterization of the first cultured oligotrophic marine Gammaproteobacterium from the SAR92 clade

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  • High-throughput culturing (HTC) consisting of extinction culturing in autoclaved seawater has led to the isolation and characterization of many novel strains of oligotrophic marine bacteria. Strain HTCC 2207 was isolated from the Oregon coast by the HTC method. Phylogenetic analysis based on 16S rRNA gene sequence showed that this strain fell into the SAR92 clade in the oligotrophic marine Gammaproteobacteria (OMG) group. The OMG group is distantly related to previously cultivated genera of Gammaproteobacteria. Initial phylogenetic characterization was followed by genome sequencing and interpretation, proteomic analysis by liquid chromatography/tandem mass spectrometry, and determination of the fatty acid profile. Culture experiments, microscopic observations, and the genome sequence indicate that HTCC 2207 cells are motile, aerobic, heterotrophic, Gram-negative, short rods of approximately 0.148 µm3. Growth characteristics were observed at six different carbon concentrations and five different temperatures. Optimal growth rate (3.15 d-1) occurred at 16 ºC in natural seawater amended with nitrogen, phosphorus, vitamins, and a mixture of organic carbon compounds yielding a maximum cell density of 1.85 × 107 cells per ml. In contrast, the maximum cell density in seawater without addition organic carbon was 1.01 × 106 cells per ml. This strain has been described previously to form small colonies on 1/10 R2A agar media, but did not growth in any other artificial media. These growth characteristics showed that HTCC 2207 is a slow growing, oligotrophic, psychro-mesophilic bacterium. Initial sequencing has so far revealed an unclosed genome of 2,619,777 base pairs coding for 2390 open reading frames. The G+C content is 49.10 mol %. The bacterium possesses all major metabolic pathways, but is requires some vitamins. Proteomic analyses identified 146 expressed proteins including a biopolymer transporter, nitrate transporter, flagellin modification proteins, urease, and a pilus assembly protein. HTCC 2207 predominantly contained the unsaturated fatty acids 18:1 ω7c and 16:1 ω7c + 16:1 ω6c. The fatty acids 16:0, 16:1, and 18:1 were commonly found in previously cultivated genera of Gammaproteobacteria. This strain also contained significant amounts of 3-OH 10:0, 3-OH 12:0, 17:1 ω8c, 14:0, and 10:0 fatty acids. From the phenotypic, genotypic, and genomic evidence, it is proposed that HTCC 2207 should be established as a new genus and species.
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