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Flexivirga Alba Gen. Nov., Sp. Nov., an Actinobacterial Taxon in the Family

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Flexivirga Alba Gen. Nov., Sp. Nov., an Actinobacterial Taxon in the Family The Journal of Antibiotics (2011) 64, 613–616 & 2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00 www.nature.com/ja ORIGINAL ARTICLE Flexivirga alba gen. nov., sp. nov., an actinobacterial taxoninthefamilyDermacoccaceae Kozue Anzai1, Tomoyasu Sugiyama2, Mayuko Sukisaki1, Yayoi Sakiyama1, Misa Otoguro1 and Katsuhiko Ando1 A novel actinobacterial strain ST13T isolated from soil near wastewater treatment facilities of an electroplating plant was subjected to a polyphasic taxonomic study. Cells of this organism were non-sporulating, and were irregular coccoid to comma shaped. The peptidoglycan of strain ST13T contained glutamic acid, serine, alanine, glycine and lysine, and represented the peptidoglycan type A4a. The whole-cell sugars contained ribose, glucose, galactose, rhamnose and mannose. The predominant menaquinone was MK-8(H4). The major fatty acid was iso-C16:0. The polar lipid contained phosphatidylglycerol. The DNA G+C content was 67.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ST13T fell within the radius of the family Dermacoccaceae, and its closest neighbor was Luteipulveratus mongoliensis MN07-A0370T (95.1%). However, strain ST13T did not make a coherent clade with members of the recognized organisms. On the basis of the phylogenetic and phenotypic characteristics of this actinobacterium, a novel genus and species, Flexivirga alba gen. nov., sp. nov., is proposed. The type strain of F. alba is ST13T (¼NBRC 107580T¼DSM 24460T). The Journal of Antibiotics (2011) 64, 613–616; doi:10.1038/ja.2011.62; published online 3 August 2011 Keywords: actinobacteria; Dermacoccaceae; Flexivirga alba gen. nov., sp. nov.; new genus INTRODUCTION incubated at 281 C for 1 week. The strain ST13T was obtained after three The family Dermacoccaceae was first proposed by Stackebrandt and successive transfers on yeast extract-soluble starch medium (YS medium; 2 g À1 Schumann,1 and its pattern of 16S rRNA signature nucleotides was yeast extract, 10 g soluble starch and 15 g l agar; pH 7.3) and maintained as emended by Zhi et al.2 At the time of writing, the family Dermacocca- pure culture at room temperature until used. For long-term preservation, strain T 1 ceae comprised of six recognized genera: Dermacoccus,3 Demetria,4 ST13 was suspended in 10% glycerol solution and stored at À80 C. Kytococcus,3 Luteipulveratus,5 Yimella6 and Branchiibius.7 The organisms belonging to these genera are Gram-positive, aerobic, non-halophilic, Morphological and physiological characteristics The cultural characteristics of strain ST13T were observed on ISP media at coccoid actinobacteria, and their peptidoglycan type is variation A4a. 281 C for 5 days. Morphological features were observed under a scanning Isolates assigned to this family are typically associated with terrestrial electron microscope (JSM-6060; JEOL, Tokyo, Japan).13 Cell motility was tested 8–11 habitats, notably, cured meat products, skin, human blood and soil. under a light microscope by observing cells suspended in phosphate buffer 14 The recently described Branchiibius hedensis was isolated from the (1 mM; pH 7.0). Gram staining was performed using Hucker’s modification. branchia of a codfish.7 During a study of aerobic bacteria from soil Growth under anaerobic conditions was determined by incubating in an T samples, we isolated a novel actinobacterium, designated ST13 ,from anaerobic chamber with an O2-absorbing and CO2-generating agent (Anae- soil near wastewater treatment facilities of an electroplating plant in ro-Pack; Mitsubishi Gas Chemical Company, INC., Tokyo, Japan). Growth tests Seki, Gifu prefecture, Japan. Phylogenetic analysis based on 16S rRNA for pH range were carried out by using media adjusted to pH 3–12 with 4 M gene sequence of strain ST13T revealed that the isolate was a member of HCl or 5 M KOH after sterilization, and NaCl tolerance was examined on YS the suborder Micrococcineae. The aim of this study was to determine the medium supplemented with 1–10% NaCl (w/v) after 1–5 days of incubation at 281 C. The optimum temperature and temperature range for growth were taxonomic position of strain ST13T by using a polyphasic approach. determined by incubating at 5, 10,15, 20, 25, 28, 37, 45 and 501 C. ISP medium 8 was used to test for nitrate reduction.15 Decomposition of urea was MATERIALS AND METHODS determined on Christensen urea agar containing 2.0% urea.16 Degradation of Isolation and cultivation casein and other compounds (final concentration 0.5%) was determined using The soil sample was collected from a depth of B2 cm from the surface near nutrient agar as the basal medium.16 Catalase activity was determined by the wastewater treatment facilities of an electroplating plant in Seki, Gifu production of bubbles after the addition of a drop of 3.0% H2O2. Oxidase prefecture, Japan. The sample was dried at room temperature for several days activity was examined using cytochrome oxidase paper (Nissui Pharmaceutical and suspended in 10 ml sterile water. The sample was vortexed, allowed to settle CO., Tokyo, Japan). Other physiological and biochemical characteristics were for 1 min, and 100 ml of the resultant solution was further diluted and spread determined by using API ZYM, API Coryne and API 50CH systems (bioMe´r- onto International Streptomyces Project (ISP) medium 2.12 The plates were ieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. 1NITE Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan and 2Tokyo University of Technology, Tokyo, Japan Correspondence: Dr M Otoguro, NITE Biological Resource Center, National Institute of Technology and Evaluation, Kazusakamatari 2-5-8, Kisarazu, Chiba 292-0818, Japan. E-mail: [email protected] Received 11 January 2011; revised 31 May 2011; accepted 15 June 2011; published online 3 August 2011 Flexivirga alba gen. nov., sp. nov. KAnzaiet al 614 Chemotaxonomic analysis Strain ST13T was subjected to chemotaxonomic analysis for its classification in the family Dermacoccaceae, including menaquinones,13 polar lipids,17 whole- cell sugars18 and DNA G+C content.19 Analysis of cellular fatty acids was carried out using the Microbial Identification System version 4.02 (MIDI, Inc., Newark, DE, USA),20 and the results were compared with the ACTINO library in the Microbial Identification System. Cell-wall amino acids were analyzed by using the method described by Tamura et al.,13 and the amino acid isomers in cell-wall peptidoglycan were analyzed using the method described by Nozawa et al.21 Biomass for chemotaxonomic studies was obtained by cultivation in shaken flasks containing YG broth (10 g glucose, 10 g yeast extract and 15 g lÀ1 agar; pH 7.3) for 24–72 h at 28 1C.22 Phylogenetic analysis DNA extraction from strain ST13T and PCR amplification of the 16S rRNA gene were performed as described by Tamura and Hatano.23 PCR product was directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an automatic DNA sequencer (ABI Prism 3730 Genetic Analyzer; Applied Biosystems). The CLUSTAL_X program24 was used to align the 16S rRNA gene sequences with related sequences (available in the GenBank/EMBL/DDBJ databases) from all of the type strains of the family Dermacoccaceae.Phylogenetictreeswere constructed by the neighbor-joining25 and maximum-likelihood26 methods using MEGA (Molecular Evolutionary Genetics Analysis) version 3.1.27 The topology of the tree was evaluated by means of bootstrap analysis based on 1000 replicates.28 Figure 1 Scanning electron micrographs of irregular coccoid or comma- shaped cells from 1-week-old cultures of strain ST13T grown on 1/2 ISP2 (a) and ISP2 (b). 0.01 Branchiibius hedensis Mer 29717T (AB520822) 85 T 77 Demetria terragena HKI 0089 (Y14152) Yimella lutea YIM 45900T (FJ528304) Luteipulveratus mongoliensis MN07-A0370T (AB468971) Flexivirga alba ST13T (AB539735) 82 Dermacoccus abyssi MT1.1T(AY894323) 100 Dermacoccus profundi MT2.2T(AY894329) 65 T 100 Dermacoccus barathri MT2.1 (AY894328) Dermacoccus nishinomiyaensis DSM20448T(X87757) Kytococcus schroeteri DSM 13884T(AJ297722) 100 Kytococcus sedentarius DSM 20547T(X87755) Streptomyces griseus subsp.griseusNBRC 12875T(AB184211) Figure 2 Phylogenetic tree based on 16S rRNA gene sequences constructed with the neighbor-joining algorithm showing the relationships between strain ST13T and members of the family Dermacoccaceae. Numbers at branch points are confidence limits estimated with bootstrap analysis based on 1000 replicates; only values of 50% are presented. Solid circles indicated that the corresponding nodes were also recovered in maximum-likelihood algorithms. Bar, 0.01 Knuc in nucleotide sequences. The Journal of Antibiotics Flexivirga alba gen. nov., sp. nov. KAnzaiet al 615 RESULTS AND DISCUSSION Morphological and physiological characteristics ) er Strain ST13T formed moist, smooth and white colonies on ISP 4 medium 2 and YS medium. The cells were aerobic, Gram-positive, 7 ), MK-8(H catalase-negative, oxidase-negative, non-sporulating and non-motile. 2 c9 c9 0.9 T 16:0 Mycelia had not developed. Strain ST13 exhibited good growth in  17:1 18:1 C C iso-C NaCl concentrations of up to 5.0% and weak growth at 6.0%. Good Rib, growth was observed at 15–37 1C, and no growth at 5 1Cand501C. The optimum temperature for growth was 25 1C. The pH range for , data from reference. growth was 5.0–9.0 and the optimum was 7.0–8.0. The strain grew well on ISP medium 2, 3, 5, 6 and 7, but grew weakly on ISP medium . Branchiibius 6 4. Scanning electron microscopy revealed near-coccoid cells (0.6– 1.3–1.8 0.7 15:0 17:0 )MK-8(H  1.2 mm in diameter) with buds on 1/2 ISP medium 2 (2 g yeast 4 15:0 extract, 5 g malt extract, 2 g dextrose and 15 g lÀ1 agar; pH 7.3) iso-C anteiso-C anteiso-C (Figure 1a).
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