Characterization of novel marine oligotrophic bacteria isolated from the Pacific Ocean : description of Marinivirgula fluito gen. nov., sp. nov., Marinivirgula obesa gen. nov., sp. nov. and Litincola parvulus gen. nov., sp. nov. Public Deposited

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  • A new high-throughput culturing (HTC) method using a low nutrient heterotrophic medium (LNHM) has led to the isolation of many novel strains of oligotrophic bacteria from marine ecosystems. Four strains belonging to a single dade, HTCC2151, HTCC218OT, HTCC2178T and HTCC2188T, were isolated from the coast of Oregon by the HTC method. Phylogenetic analysis based on their 1 6S rDNA sequences showed that they fell into the OM1 82 dade in the oligotrophic marine Gammaproteobacteria (0MG) group, which is distantly related to previously cultivated genera in Gammaproteobacteria. This analysis, along with DNA-DNA hybridization results, indicated that strains HTCC2151 and HTCC218OT were the same species, showing 89.6% genomic DNA relatedness between these strains. Strain HTCC2178T was revealed to fall into the same genus with strains HTCC2 151 and HTCC2 180T, but a different species, and this was supported by 96.5% 96.7% of 16S rRNA gene sequence similarity with the strains and 27.4% of genomic DNA relatedness between strains HTCC2 178T and HTCC218OT. Strain HTCC2188T differed significantly from HTCC2151, HTCC218OT and HTCC2178T, with less than 12.1% of the DNA-DNA hybridization result with any of the strains, and thus it was proposed that strain HTCC2188T be placed in a separate genus in the 0M182 dade. G+C mol% of strains HTCC2151, HTCC218OT, HTCC2178T and HTCC2188T were 36.2%, 41%, 54.2% and 49.3%, respectively (HPLC method). All the HTCC strains in 0M182 dade were Gram-negative, and microscopic observations revealed that they were short rods of 0.06 xm3 to 0.13 m3 in size on average and divided by binary fission. Growth curves and specific growth rates at six different temperatures and six different mixed carbon concentrations were determined. Their growth characteristics showed that these bacteria were slowly growing, obligate or facultative oligotrophic and psychro-tomesophilic bacteria. Two of the strains, HTCC2178T and HTCC2188T, started forming tiny colonies on l/1OR2A at 16°C after a 10-day incubation, but not HTCC2151 and HTCC2 180T Five different liquid artificial seawater (A SW) media were tested for their ability to support growth of the strains, and strains HTCC218OT, HTCC2 178T and HTCC2188T grew in one or two different ASW media, but not HTCC215O. pH ranges for growth of strains HTCC2151, HTCC218OT, HTCC2178T and HTCC2188T were pH 7-8.5, pH 7.5-10, pH 7-11, andpll 7.5 10. Ranges of NaCI concentrations for growth of strains HTCC2 180T, HTCC2 178T and HTCC2188Twere 1.5-3.3%, 1.0-3.3%, and2.0-3.3%. Thirty four different carbon sources were utilized by strain HTCC2 180T, fourteen by HTCC2178T and fifteen by strain HTCC21S8T as sole carbon source among forty seven different carbon sources. Strain HTCC2180T utilized all kinds of hexoses and sugar alcohols tested, most of pentoses, oligosaccharides and organic acids, some of amino acids, and one of amino sugars as sole carbon sources. Most of pentoses and hexoses, some of oligosaccharides and organic acids, and only one kind of sugar alcohols and amino acids were utilized by strain HTCC2178T, however, none of amino sugars. Strain HTCC2188T utilized most of hexoses, some of pentoses, oligosaccharides and sugar alcohols, and one of amino acids and amino sugars, but none of organic acids was utilized by this strain as sole carbon source. All the strains in the OM 182 dade showed positive reactions for alkaline phosphatase, esterase, esterase lipase and naphthol-AS-BI-phosphohydrolase. Presence of acid phosphatase and naphthol-AS-BI-phosphohydrolase was detected from strains HTCC2151 and HTCC218OT. Only one strain, HTCC2188T, exhibited positive results for leucine arylamidase, a-chymotrypsin and N-acetyl-f3- glucosaminidase. HTCC strains in the 0M182 dade contained the fatty acids, 16:0, 18:0 and 16:1, commonly found in previously cultivated genera in Gammaproteobacteria. They also showed the presence of 17:1 o7c or 17:1 w8c as one of the major fatty acids, which was not usually found as a dominant fatty acid in other genera in Gammaproteobacteria. From the genotypic and polyphasic evidences, it is proposed that strains HTCC2151, HTCC218OT and HTCC2178T should be placed into a new genus named Marinivirgula gen. nov. including Marinivirgulafluito gen. nov., sp. nov. with the type strain HTCC2 180T, and Marinivirgula obesa gen. nov., sp. nov. with the type strain HTCC2178T. The name Litincolaparvulus gen. nov., sp. nov. is suggested for the type strain HTCC2 188 T.
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