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International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3075–3078 DOI 10.1099/ijs.0.048793-0 Brevifollis gellanilyticus gen. nov., sp. nov., a gellan-gum-degrading bacterium of the phylum Verrucomicrobia Shigeto Otsuka,1 Taku Suenaga,1 Hoan Thi Vu,1 Hiroyuki Ueda,1 Akira Yokota2 and Keishi Senoo1 1 Correspondence Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Shigeto Otsuka [email protected] 2 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan The taxonomic properties of strain DC2c-G4T, a Gram-staining-negative, ovoid, gellan-gumdegrading bacterial isolate, were examined. Phylogenetic analysis based on 16S rRNA gene sequences identified this isolate as a member of the phylum Verrucomicrobia and closest to the genus Prosthecobacter. The 16S rRNA gene sequence similarities between this isolate and any of the type strains of species of the genus Prosthecobacter were less than 95 %. In addition, the absence of a single prostheca and the predominant menaquinone MK-7(H2) supported the differentiation of this isolate from the genus Prosthecobacter. Here, we propose Brevifollis gellanilyticus gen. nov., sp. nov. to accommodate the isolate. The type strain of the type species is DC2c-G4T (5NBRC 108608T5CIP 110457T). Bacteria belonging to the phylum Verrucomicrobia are recognized as being typically difficult to cultivate. Although recent advances in cultivation methods and patient efforts have gradually increased the diversity of cultured isolates and described species belonging to this phylum (e.g. Janssen et al., 2002; Sangwan et al., 2005; Yoon et al., 2007a, b, 2008, 2010), the number of cultured strains is still limited. Improved PCR techniques and metagenomic analyses are now revealing the ubiquitous distribution and even, in some cases, the dominance of members of the phylum Verrucomicrobia in the environment (Bergmann et al., 2011; Kielak et al., 2010). However, the potential difficulties in cultivation have kept the ecological functions of these organisms unknown. Therefore, it is desirable to obtain more and diverse isolates of the phylum Verrucomicrobia. In our previous study, we established consortia of Chlorella vulgaris NIES-227 (a green microalga) and bacteria originating from soil (Ueda et al., 2010). The bacterial culture fluid was collected from one of the consortia, diluted, and inoculated onto 2 % gellan gum plates containing live Chlorella prepared as previously reported (Otsuka et al., 2013). Strain DC2c-G4T was isolated after two weeks of cultivation at 30 uC, as one of the visible colonies formed on the medium. Afterward, it turned out Abbreviations: NJ, neighbour-joining; ML, maximum-likelihood. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Brevifollis gellanilyticus DC2c-G4T is AB552872. A supplementary figure is available with the online version of this paper. 048793 G 2013 IUMS that this strain grows on liquid and agar-gelled R2A (Seviour et al., 1994) and MMB (Staley & Mandel, 1973) media, but not on nutrient broth (Difco). Strain DC2c-G4T lyses gellan gum, and the surface of the gel around the colonies sagged locally when this strain was cultivated on gellan-gum-gelled media. The strain is maintained in our laboratory in glycerol stock at –80 uC. Gram staining was performed as described by Gerhardt et al. (1994). Cell morphology was observed by micrographs (Fig. 1) taken by JEOL Datum (Tokyo, Japan) using transmission electron microscopy with negative staining using the model 1010 at an acceleration voltage of 100Kv, with cells grown for 3 days or 1 week at 30 uC, namely at their middle and late exponential growth phase, on 1.5 % agar plates of R2A and MMB (separately). The bacterial cells stain Gram-negative, and are non-motile, non-gliding, single, unbranched, ovoid without prosthecae (Fig. 1) and 0.8–1.0 mm in width, 1.5–2.0 mm in length after division and up to 3.5 mm in length shortly before division. Without DNA extraction, the almost full-length 16S rRNA gene was amplified by PCR with the primers 27F and 1492R (Lane, 1991) by the same method and reaction conditions as described elsewhere (Otsuka et al., 2008), with Biotaq DNA polymerase (Bioline) and a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems). The amplified products were subjected to sequencing commercially performed by Takara Bio (Mie, Japan). The determined 16S rRNA gene sequence of strain DC2c-G4T was a continuous stretch of 1469 bp. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 09:10:06 Printed in Great Britain 3075 S. Otsuka and others was checked, and distances (according to the Kimura twoparameter model) and clustering with the maximumlikelihood (ML) and neighbour-joining (NJ) methods were determined by using bootstrap values based on 1000 replications. In the ML and NJ trees (Fig. 2 and Fig. S1 available in IJSEM Online, respectively), strain DC2c-G4T was located within the cluster including all species of the genus Prosthecobacter (indicated as ‘Prosthecobacter cluster’ in the trees). Fig. 1. Transmission electron micrograph of negatively stained cells of strain DC2c-G4T. Bar, 1 mm. The close relatives of strain DC2c-G4T among species with validly published names were searched for as described elsewhere (Vu et al., 2010). Species of the genus Prosthecobacter were the closest taxa, with sequence similarities of 92.9–94.9 % calculated by EMBOSS Matcher (Huang & Miller, 1991). These values are low enough to describe a novel species without a DNA–DNA hybridization experiment (Stackebrandt & Goebel, 1994) or even a new genus (Martinez-Romero & Caballero-Mellado, 1996) represented by strain DC2c-G4T. The 16S rRNA gene sequences were aligned based on secondary structure using the SILVA SINA Webaligner (Pruesse et al., 2007) and imported into MEGA 5 (Kumar et al., 2008). The alignment 0.05 50/70 84 /87 The respiratory quinone system and cellular fatty acid composition of strain DC2c-G4T after 3 days of cultivation at 30 uC on R2A agar medium were analysed according to the same conditions described by Katsuta et al. (2005), by using a GC (GC6890; Hewlett Packard) equipped with a 25 m60.2 mm Ultra 2 capillary column (19091B-102E; Agient). The fatty acids were identified with MIDI Sherlock Version 4.0 and the TSBA40 database. The G+C content of the chromosomal DNA was determined, as described by Mesbah et al. (1989), using reversed-phase HPLC. Menaquinone MK-7(H2) was detected as the predominant quinone. The major fatty acids were iso-C14 : 0 (27.1 %), C16 : 0 (27.0 %), C16 : 1v11c (10.4 %), C16 : 1v5c (8.4 %), C14 : 0 (6.5 %), anteiso-C15 : 0 (6.1 %) and C15 : 0 (4.9 %). The DNA G+C content of strain DC2c-G4T was 59.4 mol%. Growth under anaerobic conditions was tested by cultivating strain DC2c-G4T in an anaerobic chamber with an O2-absorbing and CO2-generating agent (AnaeroPack; Mitsubishi Gas Chemical). Salinity and pH ranges for growth were tested by incubating the strain for a week at Brevifollis gellanilyticus DC2c-G4T (AB552872) Prosthecobacter fusiformis FC4T (U60015) Prosthecobacter dejongeii FC1T (U60012) 99 Prosthecobacter debontii FC3T (U60014) /100 Prosthecobacter vanneervenii FC2T (U60013) 98/100 Prosthecobacter fluviatilis JCM 14805T (AB305640) 100/100 100/100 ‘Prosthecobacter cluster’ Verrucomicrobium spinosum DSM 4136T (X90515) 96/96 43/– Roseimicrobium gellanilyticum DC2a-G7T (AB552861) Akkermansia muciniphila ATCC BAA-835T (AY271254) 91/94 Luteolibacter pohnpeiensis KCTC 22041T (AB331895) 82/85 Haloferula rosea KCTC 22201T (AB372853) Roseibacillus ishigakijimensis KCTC 12986T (AB331888) 99/99 93/89 97/98 Rubritalea marina DSM 17716T (DQ302104) Persicirhabdus sediminis KCTC 22039T (AB331886) Opitutus terrae JCM 15787T (AJ229235) Chlamydia trachomatis ATCC VR-571BT (D89067) Fig. 2. ML tree reconstructed based on the nearly full-length 16S rRNA gene sequences of strain DC2c-G4T and the type strains of phylogenetically related species. Chlamydia trachomatis ATCC VR-571BT was used as the outgroup. Accession numbers in DDBJ/EMBL/GenBank databases are indicated in parentheses. Bootstrap values based on 1000 replications for ML and NJ analyses are indicated at the corresponding nodes (ML/NJ). Tree topologies differed between ML and NJ trees at the node indicated by an open circle. Bar, 0.05 nucleotide substitutions per site. 3076 Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 63 IP: 88.99.165.207 On: Sun, 30 Apr 2017 09:10:06 Brevifollis gellanilyticus gen. nov., sp. nov. 30 uC in R2A medium supplemented with 0, 1, 4, 8, 9, or 10 % NaCl (w/v) and in R2A medium adjusted to pH 5 to 10 at 1 pH unit intervals using HCl or NaOH. Tolerance of antibiotics was examined in three repetitions by Biolog Gen III MicroPlate during 1 week of incubation at 30 uC. This period of time was necessary for the positive control to become clearly positive. Growth on saccharides, catalase activity, oxidase activity and the temperature range for growth were determined as previously described (Otsuka et al., 2013). The results are given in the species description. As described above, strain DC2c-G4T can be differentiated from members of the genus Prosthecobacter by the menaquinone profile and the absence of prostheca (Table 1), although the strain is located within the ‘Prosthecobacter cluster’ in the phylogenetic trees (Figs 2 and S1). In order to describe a novel species of the genus Prosthecobacter represented by strain DC2c-G4T, we would need to emend the genus description to remove the distinguishing characteristic of this genus, the presence of a single prostheca, which may cause confusion. Alternatively, if we describe a new genus represented by strain DC2c-G4T, we should admit the presence of a different genus within the ‘Prosthecobacter cluster’ in the phylogenetic trees (Figs 2 and S1). Considering that a new taxon can arise from an already existing taxon, it is not awkward if the ‘Prosthecobacter cluster’ embeds a different genus as a result of evolutionary differentiation. Therefore, we propose that strain DC2c-G4T represents a novel species in a new genus. Description of Brevifollis gen. nov. Brevifollis (Bre.vi.fol9lis. L. adj. brevis short, small; L. masc. n. follis a windball; N.L. masc. n. Brevifollis a short ball, intended to mean a small ovoid cell). Cells are ovoid to short fusiform, single, unbranched, non– motile, non–gliding and stain Gram-negative. Obligately aerobic. Chemo-organotrophic. The major respiratory quinone is menaquinone MK-7(H2). The DNA G+C content is approximately 59–60 mol%. The type species is Brevifollis gellanilyticus. The differentiation of this genus from the phylogenetically close genus Prosthecobacter is possible chiefly by the major menaquinone MK-7(H2) and the absence of a single polar prostheca. The type species is Brevifollis gellanilyticus. Description of Brevifollis gellanilyticus sp. nov. Brevifollis gellanilyticus [gel.lan.i.ly9ti.cus. N.L. neut. n. gellanum gellan; N.L. masc. adj. lyticus (from Gr. adj. lutikon) able to loose, able to dissolve; N.L. masc. adj. gellanilyticus gellan-dissolving]. Cells stain Gram-negative and are non-motile, non-gliding, single, unbranched, ovoid to short fusiform and 0.8–162– 3.5 mm in diameter. Forms opaque pale-yellow colonies with a smooth, convex and circular surface after 3 days to 1 week of cultivation at 30 uC on R2A agar and MMB agar media. The predominant respiratory quinone is menaquinone MK7(H2). The major fatty acids are iso-C14 : 0 and C16 : 0. Grows well at 26 to 30 uC, can grow at 10 to 37 uC, but does not grow or hardly grows at 4 or 42 uC; grows in media at pH 6 to 8 but not at pH 5 or 9; can tolerate 8 % but not 9 % salinity. Sensitive to tetrazolium violet and tetrazolium blue. Insensitive to 1 % sodium lactate, troleandomycin, lincomycin, vancomycin, nalidixic acid, aztreonam, fusidic acid, D-serine, rifamycin SV, minocycline, guanidine HCl, lithium chloride, potassium tellurite, sodium butyrate and sodium bromate. Growth occurs on D-glucose, D-galactose, Dfructose (slow growth), L-rhamnose, D-mannose, L-xylose, Table 1. Characteristics differentiating strain DC2c-G4T from phylogenetically close genera Taxa: 1, DC2c-G4T; 2, Prosthecobacter; 3, Verrucomicrobium; 4, Roseimicrobium. Data for Prosthecobacter, Verrucomicrobium and Roseimicrobium from Hedlund et al. (1997), Hedlund (2010), Otsuka et al. (2013) & Takeda et al. (2008). +, Positive; –, negative; NA, data not available. Characteristic Cell shape Cell length Cell width Multiple short prosthecae Single polar prostheca Colony colour Optimal growth temperature (uC) pH range for growth Salinity tolerance (% NaCl) Aerobic growth Anaerobic growth Major menaquinones DNA G+C content (mol%) 1 2 3 Ovoid to short fusiform 2–3.5 0.8–1 – – Pale yellow 10–37 6–8 8 + – MK-7(H2) 59.4 Fusiform to vibrioid 2–10 0.5–1 – + Pale yellow 1–40 6.5–7* Rod 1–3.8 0.8–1 + – Pale yellow 26–33 NA + + MK-6(H2) 54.6–62.9 4 Ovoid to rod 2–4.5 0.9–1.5 – – Orangish-red 26–30 NA 6–7.5 1 1 + + – – MK-9, MK-10, MK-10(H2) MK-9, MK-10, MK-11 58–59 60.1 *pH range for growth of Prosthecobacter is that of Prosthecobacter fluviatilis HAQ-1T (Takeda et al., 2008). http://ijs.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 09:10:06 3077 S. Otsuka and others D-sorbitol, D-gluconic acid, sucrose, starch, pullulan, inulin, curdlan, gellan gum, xanthan gum, galactomannan and xylan (very slow growth). No growth occurs on Dglucuronic acid, glycerol, pyruvate, cellulose (soluble) or alginate. Positive for cytochrome oxidase and catalase. Otsuka, S., Sudiana, I.-M., Komori, A., Isobe, K., Deguchi, S., Nishiyama, M., Shimizu, H. & Senoo, K. (2008). Community The type strain is DC2c-G4T (5NBRC 108608T5CIP 110457T), isolated from an artificial consortium of Chlorella vulgaris (green alga) and a mixed population of bacteria originating from soil. The DNA G+C content of strain DC2c-G4T is 59.4 mol%. nov., a new member of the class Verrucomicrobiae. Int J Syst Evol Microbiol 63, 1982–1986. structure of soil bacteria in a tropical rainforest several years after fire. Microbes Environ 23, 49–56. Otsuka, S., Ueda, H., Suenaga, T., Uchino, Y., Hamada, M., Yokota, A. & Senoo, K. (2013). Roseimicrobium gellanilyticum gen. nov., sp. 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