International Journal of Systematic and Evolutionary Microbiology (2016), 66, 5412–5416 DOI 10.1099/ijsem.0.001533 Carboxylicivirga flava sp. nov., isolated from marine surface sediment Hui Wang,1 Cancan Qi,1 Weiwei Chen,1 Wenwen Dong,1 Haitian Tang2 and Xiaoke Hu1 Correspondence 1Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Costal Zone Xiaoke Hu Research, Chinese Academy of Sciences, Yantai 264003, PR China [email protected] 2Yantai Marine Environment Monitoring Central Station, State Oceanic Administration, Yantai 264006, PR China A novel bacterial strain, designated Q15T, was isolated from sediments obtained from the Bohai Sea in China and subjected to a polyphasic taxonomic study. Cells of strain Q15T were Gram- stain-negative, strictly aerobic rods that produced circular, flat, orange colonies. Phylogenetic analysis based on 16S rRNA gene sequences revealed that Q15T was affiliated with the genus Carboxylicivirga in the family Marinilabiliaceae of the phylum Bacteroidetes. Strain Q15T differed genotypically from the type strains of the three recognized species of this genus (Carboxylicivirga taeanensis MEBiC 08903T, Carboxylicivirga mesophila MEBiC 07026T and Carboxylicivirga linearis FB218T) and shared 94.0–95.2 % 16S rRNA gene sequence similarity with them. The DNA G+C content of strain Q15T was 44.7 mol%. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0 3-OH, and menaquinone MK-7 was the main respiratory quinone. Polar lipids contained phosphatidylethanolamine, an unidentified aminolipid, an unidentified phospholipid and other unknown lipids. Based on the data from the current polyphasic analysis, a novel species, Carboxylicivirga flava sp. nov., is hereby proposed with Q15T (=CICC 23923T=KCTC 42707T) as the type strain. The family Marinilabiliaceae (Ludwig et al., 2011) includes, from Daebudo-Island (126 269¢ E 37 169¢ N) and Taean the time of writing, 10 genera: Alkaliflexus (Zhilina et al., County (126 119¢ E 36 499¢ N), Korea, respectively, and 2004), Alkalitalea (Zhao & Chen, 2012), Anaerophaga (Denger C. linearis FB218T was isolated from Rongcheng, China (122 et al., 2002), Carboxylicivirga (Yang et al., 2014), Geofilum 14¢ 34† E 36 54¢ 36† N). Herein, a novel strain, Q15T, was (Miyazaki et al., 2012), Mangroviflexus (Zhao et al., 2012), isolated from the sediment in the same region and proposed Marinilabilia (Nakagawa & Yamasato, 1996), Natronoflexus to represent a novel species in the genus. The novel bacterial (Sorokin et al., 2011), Saccharicrinis (Yang et al., 2014) and strain was isolated during investigation into the succession of Thermophagus (Gao et al., 2013); and one misclassified spe- bacteria during the process of biodegradation of different cies: [Cytophaga] xylanolytica (Haack & Breznak, 1993). The types of hydrocarbons. Sediments for these studies were col- genus Carboxylicivirga was first proposed by Yang et al. (2014) lected from the Bohai Sea (37 40¢ 20† N 120 20¢ 30† E), comprising two species, Carboxylicivirga mesophila and northern China. Subsurface sediments (5 cm beneath the sur- Carboxylicivirga taeanensis. A recent study identified a third face) were kept at 4 C and immediately transferred to the lab- species in the genus Carboxylicivirga, namely Carboxylicivirga oratory for further analysis. Aliquots (10 g) of the collected linearis (Wang et al., 2015). Interestingly, these three species sediments were inoculated into 100 ml of Difco marine broth were all isolated from marine environments. C. taeanensis T T 2216 medium (MB; BD Bioscience) and incubated at MEBiC 08903 and C. mesophila MEBiC 07026 were isolated 180 r.p.m. and 25 C for 48 h. The liquid cultures were serially diluted and spread on Difco marine agar 2216 plates (MA; BD T Abbreviations: ME, minimum-evolution; ML, maximum-likelihood; NJ, Bioscience). After incubation at 25 C for 2 days, strain Q15 neighbour-joining. was successfully isolated and cryo-preserved at À80 C in MB The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene supplemented with 30 % (v/v) glycerol. Polyphasic tests sequence of Carboxylicivirga flava Q15T is KR809872. including phylogenetic, genotypic, chemotaxonomic and Three supplementary figures and three supplementary tables are avail- other phenotypic assays were performed to identify the the T able with the online Supplementary Material. taxonomic position of strain Q15 . Downloaded from www.microbiologyresearch.org by 5412 001533 ã 2016 IUMS Printed in Great Britain IP: 130.39.51.49 On: Wed, 08 Feb 2017 16:44:13 Carboxylicivirga flava sp. nov. After incubation on MA for 72 h at 30 C under aerobic server (Kim et al., 2012). The most closely related strains to conditions, strain Q15T strain formed circular, flat, orange Q15T and strains of selected genera belonging to the family colonies with approximate diameters of 0.5–1.0 mm. The Marinilabiliaceae in the phylum Bacteroidetes were subjected Gram reaction was tested according to the method to phylogenetic analysis. Phylogenetic trees were recon- described by Gerhardt et al. (1994). Cell morphology and structed using the maximum-likelihood (ML), neighbour- motility was examined by using scanning electron micros- joining (NJ) and minimum-evolution (ME) algorithms copy after 24 h of incubation in MB. Growth under anaero- using the MEGA 5 software package (Tamura et al., 2011). bic conditions was examined on MA supplemented with The robustness of the inferred tree topologies was evaluated 0.5 % acetate and 0.25 % NaNO3 (both w/v) at 30 C in an after 1000 bootstrap replicates of the ML, NJ and ME data, anaerobic chamber as described by Yakimov et al. (2003). respectively. The analysis of 16S rRNA gene sequences using The results showed that strain Q15T was a Gram-stain- the EzTaxon server indicated that strain Q15T showed high- negative, rod-shaped (0.3–0.6Â2.5–18.7 µm) bacterium est similarity to members of the genus Carboxylicivirga in (Fig. S1, available in the online Supplementary Material). the family Marinilabiliaceae. C. taeanensis MEBiC 08903T, Flagellum and gliding motility were not observed. C. mesophila MEBiC 07026T and C. linearis FB218T exhib- ited 95.22, 94.31 and 94.00 % 16S rRNA gene sequence sim- The genomic DNA of Q15T strain and previously character- ilarity to the novel strain, respectively, with all others ized strains in the genus Carboxylicivirga (C. taeanensis sharing less than 94.00 %. The ML, NJ and ME algorithms MEBiC 08903T, C. mesophila MEBiC 07026T and C. linearis generated similar phylogenetic trees [Fig. 1 (ML) and Fig. FB218T) were extracted using the commercial Ultra-Clean S2 (NJ and ME)]. Phylogenetic analysis of strain Q15T microbial DNA isolation kit (MoBio Laboratories). C. taea- based on the ML tree indicated that it grouped with C. taea- nensis MEBiC 08903T and C. mesophila MEBiC 07026T nensis MEBiC 08903T, C. mesophila MEBiC 07026T and strains were purchased from the Japan Collection of Micro- C. linearis FB218T, and was distinguished from all other organisms (JCM), and C. linearis FB218T was kindly pro- type strains in the family. This indicated that strain Q15T vided by Dr Zongjun Du from the College of Marine was genotypically different from the three identified species Science, Shandong University at Weihai. High-quality geno- in the genus Carboxylicivirga and represented a novel mic DNA was used for PCR amplification by using the 16S species. rRNA gene primers 27F and 1492R (Wang et al., 2012). Amplicons were sequenced for the almost full-length 16S Chemotaxonomic and phenotypic characterizations were rRNA gene, which was then analysed using the EzTaxon conducted on strain Q15T simultaneously with reference Alkaliflexus imshenetskii Z-7010T (NR117198) 87 Geofilum rubicundum JAM-BA0501 T (NR112717) 64 Mangroviflexus xiamenensis P2 T (NR109309) 0.02 87 Cytophaga xylanolytica DSM 6779 T (NR117112) Alkalitalea saponilacus SC/BZ-SP2 T (NR117932) 80 99 Natronoflexus pectinivorans AP1 T (NR108635) Marinilabilia salmonicolor NCIMB 2216 T (NR115480) 99 Anaerophaga thermohalophila Fru22 T (NR028963) 99 Thermophagus xiamenensis HS1 T (NR108181) Saccharicrinis fermentans ATCC 19072 T (M58766) 100 Saccharicrinis marinus CICC 10837 T (KJ093446) Saccharicrinis carchari CICC 10590T (JQ683776) 89 Carboxylicivirga flava Q15T (KR809872) T 99 Carboxylicivirga taeanensis MEBiC 08903 (KF620113) T 83 Carboxylicivirga linearis FB218 (KP172527) 67 Carboxylicivirga mesophila MEBiC 07026T (JQ672625) Porphyromonas asaccharolytica JCM 6326 T (NR113079) Fig. 1. ML tree based on partial 16S rRNA gene sequences (1189 bp) of strain Q15T and representative members of selected genera belonging to the family Marinilabiliaceae in the phylum Bacteroidetes. The tree was reconstructed by using T MEGA 5 software. Porphyromonas asaccharolytica JCM 6326 (GenBank accession number NR113079) was used as the out- group. Bootstrap confidence values >60 % are shown at nodes. Bar, 0.02 substitutions per nucleotide position. Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 5413 IP: 130.39.51.49 On: Wed, 08 Feb 2017 16:44:13 H. Wang and others strains C. taeanensis MEBiC 08903T, C. mesophila MEBiC biochemical characteristics are provided with other species 07026T and C. linearis FB218T. Growth at varying tempera- descriptive parameters in Tables 1 and S1. The novel strain tures (15, 20, 25, 30, 37, 45 and 50 C), pH (4, 5, 6, 7, 8, 9, 10 Q15T showed high sensitivity to all 10 tested antibiotics, and 11) and NaCl concentrations (0, 2,