Rubrivirga marina gen. nov., sp. nov., a member of the family Rhodothermaceae isolated from deep seawater

Two aerobic, Gram-stain-negative, pale-red-pigmented and rod-shaped bacterial strains, designated SAORIC-26 and SAORIC-28, were isolated from seawater (3000 m depth) from the Pacific Ocean. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that the novel isolates could be affiliated with the family Rhodothermaceae of the class Cytophagia. Strains SAORIC-26 and SAORIC-28 shared 99.7 % pairwise sequence similarity with each other and showed less than 92.6 % similarity with other cultivated members of the class Cytophagia. The strains were found to be non-motile, oxidase-positive, catalase-negative and able to hydrolyse gelatin and aesculin. The DNA G+C contents were determined to be 64.8– 65.8 mol% and MK-7 was the predominant menaquinone. Summed feature 9 (iso-C17 : 1v9c and/ or C16 : 0 10-methyl), summed feature 3 (C16 : 1v6c and/or C16 : 1v7c) and iso-C15 : 0 were found to be the major cellular fatty acids. On the basis of this taxonomic study using a polyphasic approach, it was concluded that strains SAORIC-26 and SAORIC-28 represent a novel species of a new genus in the family Rhodothermaceae, for which the name Rubrivirga marina gen. nov., sp. nov. is proposed. The type strain of the type species of is SAORIC-28 (5KCTC 238675NBRC 108816). An additional strain of the species is SAORIC-26.

The recently validated family Rhodothermaceae (Ludwig et al., 2011) belonging to the class Cytophagia in the phylum Bacteroidetes comprises four genera, Rhodothermus (Alfredsson et al., 1988), Salinibacter (Anto ´n et al., 2002) Salisaeta (Vaisman & Oren, 2009) and Rubricoccus (Park et al., 2011).Three genera Rhodothermus, Salinibacter and Salisaeta have been isolated from extreme environments and exhibit thermophilic or halophilic characteristics.At the time of writing the genus Rubricoccus contains only one species with a validly published name, which was, in contrast to members of the other three genera, isolated from a euphotic zone of the Pacific Ocean and showed mesophilic and slightly halophilic characteristics.In this study we isolated two novel bacteria from deep seawater and investigated their biochemical and physiological characteristics to determine their taxonomic status.v) NaCl.Gram-staining was performed as described by Murray et al. (1994).Cell morphology and motility were observed by light microscopy (E600; Nikon) and transmission electron microscopy (TEM).Gliding motility was determined as described by Perry (1973).Growth under anaerobic conditions was determined after incubation for 4 weeks in an AnaeroPack (Mitsubishi Gas Chemical) on 1/2 MA supplemented with 1 % NaCl.Catalase activity was determined by bubble formation in a 3 % H 2 O 2 solution.Oxidase activity was determined using cytochrome oxidase test paper (Nissui Pharmaceutical).API 20E, API 20NE, API 50CH and API ZYM strips (bioMe ´rieux) were used to determine the physiological and biochemical characteristics.All suspension media for the API test strips were supplemented with 2 % (w/v) NaCl solution (final concentration).The results of API 20E, API 20 NE and API 50 CH tests strips were recorded after 5 days and those of API ZYM were recorded after 2 days at 30 u C. Determination of the respiratory quinone was carried out as described previously (Xie & Yokota, 2003).Bacterial strains grown on 1/2 MA supplemented with 1 % NaCl for 10 days at 30 u C was used for the analysis of fatty acid methyl esters.Fatty acid methyl esters were extracted and prepared according to standard protocols provided by the MIDI/Hewlett Packard Microbial Identification system (Sasser, 1990) using MIDI version 6.0 with TSBA6 database.Polar lipids were extracted according to the procedures described by Minnikin et al. (1984).They were identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984;Komagata & Suzuki, 1987).Phospholipids were detected with the Zinzadze reagent of Dittmer & Lester (1964).Whole-lipid profiles were detected by spraying with molybdatophosphoric acid (5 g molybdatophosphoric acid hydrate in 100 ml ethanol) followed by heating at 150 u C (Worliczek et al., 2007).DNA was prepared according to the method of Marmur (1961) from cells grown on 1/2 MA supplemented with 1 % NaCl and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989).A fragment of approximately 1450 bp from the 16S rRNA gene was amplified from the extracted DNA by using bacterial universal primers 27F and 1492R (Lane, 1991: Weisburg et al., 1991).To ascertain the phylogenetic position of the novel isolate, the 16S rRNA gene sequences of strains SAORIC-26 and SAORIC-28 T were compared with sequences obtained from GenBank (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov).Multiple alignments of sequences were performed using CLUSTAL_X (version 1.83) (Thompson et al., 1997) and gaps and ambiguous bases were omitted for sequence comparison.Aligned sequences were analysed using MEGA  version 4 (Tamura et al., 2007).Evolutionary distances were calculated using distance options according to the Kimura two-parameter model (Kimura, 1983).Clustering with the neighbour-joining (NJ) (Saitou & Nei, 1987) and maximum-likelihood (ML) (Felsenstein, 1981) methods was performed using ML version 2.4.4 (Guindon & Gasceul, 2003).Bootstrap values (Felsenstein, 1985) were calculated from 1000 replications.Pair-wise sequence similarities were calculated using the EzTaxon-e server (Kim et al., 2012).
The almost complete 16S rRNA gene sequence for strain SAORIC-26 and SAORIC-28 T were determined and BLAST search results with the GenBank database showed that these strains belong to the family Rhodothermaceae.Phylogenetic analyses of 16S rRNA gene sequences showed that these strains formed a cluster with Rubricoccus marinus SG-29 T with bootstrap confidence values of 100 % (NJ and ML) (Fig. 1).In particular, strains SAORIC-26 and SAORIC-28 T showed 99.7 % sequence similarity with each other and displayed 92.6 % similarity with Rubricoccus marinus SG-29 T , 86.4 % and 86.5 % sequence similarity, respectively with Salinibacter iranicus CB7 T and 86.2 % and 86.3 % sequence similarity, respectively with Salinibacter luteus DGO T .Therefore, on the basis of these phylogenetic data, strains SAORIC-26 and SAORIC-28 T should be placed into a novel species of a new genus within the family Rhodothermaceae.
Phenotypic differences among strains SAORIC-26, SAORIC-28 T and members of related genera are shown in Tables 1 and 2. These accumulated data showed that strains SAORIC-26 and SAORIC-28 T  Rubrivirga marina exhibits the following properties in addition to those given in the genus description.Colonies are circular and 1-2 mm in diameter after 10 days incubation of 1/2 MA supplemented with 1 % NaCl.
The type strain, SAROIC-28 T (5KCTC 23867 T 5NBRC 108816 T ), was isolated from deep seawater (depth; 3000 m) from the western North Pacific Ocean near Japan.The DNA G+C content of the type strain is 68.9 mol%.Strain SAORIC-26 (5KCTC 238665NBRC 108815), is an additional strain of the species.
*Summed features represent groups of two or three fatty acids that cannot be separated by the Microbial Identification System.Summed feature 3 comprises C 16 : 1 v6c and/or C 16 : 1 v7c; summed feature 9 comprises iso-C 17 : 1 v9c and/or C 16 : 0 10-methyl.