International Journal of Systematic and Evolutionary Microbiology (2013), 63, 141–145 DOI 10.1099/ijs.0.040196-0

Pseudovibrio axinellae sp. nov., isolated from an Irish marine sponge

John A. O’Halloran,1,2 Teresa M. Barbosa,2 John P. Morrissey,2 Jonathan Kennedy,3 Alan D. W. Dobson2,3 and Fergal O’Gara1,2

Correspondence 1BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland Fergal O’Gara 2Department of Microbiology, University College Cork, Cork, Ireland [email protected] 3Marine Biotechnology Centre, Environmental Research Institute, University College Cork, Cork, Ireland

A Gram-negative, motile, rod-shaped bacterial strain, designated Ad2T, was isolated from a marine sponge, Axinella dissimilis, which was collected from a semi-enclosed marine lake in Ireland. Strain Ad2T grew optimally at 24 6C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ad2T clustered with members of the genus Pseudovibrio, and showed 97.3–98.2 % sequence similarity to the type strains of recognized Pseudovibrio species. DNA–DNA relatedness values between strain Ad2T and the type strains of other Pseudovibrio species were ,27 %. The DNA G+C content of strain Ad2T was 50.5 mol%. The major fatty acid was 18 : 1v7c. Differences in phenotypic properties, together with phylogenetic and DNA–DNA hybridization analyses, indicated that strain Ad2T represented a novel species of the genus Pseudovibrio. The name Pseudovibrio axinellae sp. nov. is proposed, with Ad2T (5DSM 24994T5NCIMB 14761T) as the type strain.

The genus Pseudovibrio was first described by Shieh et al. based on 16S rRNA gene sequencing and randomly amplified (2004) and, at the time of writing, comprised three species: polymorphic DNA analysis. Here, we report on the charac- Pseudovibrio denitrificans (Shieh et al., 2004) and terization of strain Ad2T using a polyphasic approach. Pseudovibrio japonicus (Hosoya & Yokota, 2007), whose Strain Ad2T was isolated on SYP-SW agar [1 % (w/v) type strains were isolated from seawater, and Pseudovibrio starch, 0.4 % (w/v) yeast extract, 0.2 % (w/v) peptone, ascidiaceicola (Fukunaga et al., 2006), whose type strain was 3.33 % (w/v) artificial sea salts (Instant Ocean), 1.5 % (w/v) isolated from a sea squirt (ascidian). In the search for agar] as previously described (O’Halloran et al., 2011) and clinically relevant antimicrobial compounds, members of was routinely maintained on SYP-SW agar at 24 uC, as the genus Pseudovibrio are of interest due to their bioactive were the three reference strains P. denitrificans DSM potential (Kennedy et al., 2009; Santos et al., 2010; T T 17465 , P. japonicus NCIMB 14279 and P. ascidiaceicola O’Halloran et al., 2011; Flemer et al., 2012), and a number DSM 16392T, obtained from the German Collection of of antimicrobial compounds from members of the genus Microorganisms and Cell Cultures (DSMZ) or the Pseudovibrio have been characterized (Sertan-de Guzman National Collection of Industrial, Food, and Marine et al., 2007; Penesyan et al., 2011; Vizcaino, 2011). Bacteria (NCIMB). Members of the genus Pseudovibrio are frequently isolated from sponges (Hentschel et al., 2001; Webster & Hill, 2001; Colonies of strain Ad2T were circular, smooth and beige in Thiel & Imhoff, 2003; Enticknap et al., 2006; Muscholl- colour with an entire margin on SYP-SW agar. Phase- Silberhorn et al., 2008; Kennedy et al., 2009; Menezes et al., contrast microscopy of early stationary phase cells of strain T 2010; Santos et al., 2010) and we have previously analysed a Ad2 grown in SYP-SW broth at 24 uC revealed that the population of Pseudovibrio species isolated from a number cells were rod-shaped and motile, 2.0–3.5 mm long by 0.7– of marine sponges (O’Halloran et al., 2011). One of the 1.0 mm wide. Transmission electron microscopy of cells of T isolates, Ad2T, which displayed narrow-spectrum antimi- strain Ad2 negatively stained with uranyl acetate showed crobial activity, appeared to represent a distinct species that, similarly to the previously reported type strains of species of the genus Pseudovibrio, strain Ad2T cells possessed one to several subpolar flagella (Fig. S1, available The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene in IJSEM Online). sequence of strain Ad2T is JN167515. Two supplementary tables and two supplementary figures are available For phylogenetic analysis, DNA was extracted from strain T with the online version of this paper. Ad2 using an UltraClean microbial DNA isolation kit

Downloaded from www.microbiologyresearch.org by 040196 G 2013 IUMS Printed in Great Britain 141 IP: 54.70.40.11 On: Fri, 30 Nov 2018 17:58:27 J. A. O’Halloran and others

(MoBio) according to the manufacturer’s protocol. To DSMZ, Braunschweig, Germany, where cells were dis- amplify the 16S rRNA gene, the universal primers 27f (59- rupted using a French press and the DNA in the crude AGAGTTTGATCMTGGCTCAG-39) and 1492r (59-TACG- lysate was purified according to the method of Cashion GYTACCTTGTTACGACTT-39) were used (Lane, 1991) et al. (1977). DNA–DNA hybridization was performed in with a thermal cycling programme of 95 uC for 5 min, duplicate and carried out as described by De Ley et al. followed by 30 cycles of 95 uC for 30 s, 50 uC for 30 s and (1970) under consideration of the modifications described 72 uC for 90 s, and a final elongation step of 72 uC for by Huss et al. (1983). Strain Ad2T showed low DNA–DNA 7 min. The amplification product was purified using a hybridization values to P. ascidiaceicola DSM 16392T, P. QIAquick PCR purification kit (Qiagen), cloned using a denitrificans DSM 17465T, and P. japonicus NCIMB TOPO TA cloning kit (Invitrogen), and sequenced by 14279T, with average values of 26.1, 18.1 and 15.7 %, GATC Biotech using the M13 forward (59-GTAAA- respectively. These values are far below the recommended ACGACGGCCAG-39) and M13 reverse (59-CAGGAAA- threshold value of 70 % for delineation of bacterial species CAGCTATGAC-39) primers. The nearly complete (Wayne et al., 1987). The DNA G+C content of strain (1446 bp) sequence of the 16S rRNA gene of strain Ad2T Ad2T was 50.5 mol% according to the method of Mesbah was compared with sequences in the GenBank database et al. (1989). using BLAST (Altschul et al., 1997) and aligned with The cellular fatty acid contents of strain Ad2T and reference 16S rRNA gene sequences using CLUSTAL X Pseudovibrio reference strains were analysed using the (Thompson et al., 1997). Phylogenetic analyses were Microbial Identification system (MIDI) and carried out carried out using the neighbour-joining (Saitou & Nei, by the DSMZ, where fatty acid methyl esters were obtained 1987) and maximum-parsimony (Fitch, 1971) methods from 40 mg of cells grown for 24 h at 28 uC on marine using MEGA4 (Tamura et al., 2007). Bootstrap tests agar (Difco) by saponification, methylation and extraction (Felsenstein, 1985) were performed 1000 times. The according to the methods of Miller (1982) and Kuykendall neighbour-joining phylogenetic analysis indicated that et al. (1988). Similarly to virtually all members of the strain Ad2T was a member of the genus Pseudovibrio Alphaproteobacteria, the major fatty acid of strain Ad2T (Fig. 1), and this was supported by the maximum- was 18 : 1v7c. In contrast to the three Pseudovibrio parsimony phylogenetic analysis, where a similar tree reference strains, 16 : 0 and summed feature 3 (15 : 0 iso topology was observed (Fig. S2). The levels of 16S rRNA 2-OH and/or 16 : 1v7c) also represented a significant gene sequence similarity between strain Ad2T and P. proportion (.10 %) of the cellular fatty acids in strain ascidiaceicola F423T, P. denitrificans DN34T and P. Ad2T, while 15 : 0 and 17 : 0 were also detected in strain japonicus WSF2T were calculated using the EzTaxon-e tool Ad2T but not in the reference strains. Detailed informa- (Kim et al., 2012) as 98.2, 97.3 and 98.3 %, respectively. tion on the cellular fatty acid composition of strain DNA–DNA hybridization and determination of the DNA Ad2T and Pseudovibrio reference strains is provided in G+C content of strain Ad2T were carried out by the Table S1.

53 Labrenzia alba CECT 5095T (AJ878875) 90 Labrenzia aggregata IAM 12614T (D88520) 88 Labrenzia marina mano18T (AY628423) Labrenzia alexandrii DFL-11T (AJ582083) 0.01 T 77 Roseibium hamelinense OCh 368 (D85836) 96 Roseibium denhamense OCh 254T (D85832) 89 Pannonibacter phragmitetus C6/19T (AJ400704) Nesiotobacter exalbescens LA33BT (AF513441) DN34T (NR_029112) 86 Pseudovibrio denitrificans 84 Pseudovibrio axinellae Ad2T (JN167515) 100 T 63 Pseudovibrio japonicus WSF2 (AB246748) 92 Pseudovibrio ascidiaceicola F423T (AB175663) Stappia stellulata IAM 12621T (D88525) Ahrensia kielensis IAM 12618T (D88524) Ruegeria atlantica NBRC 15792T (AB255399) 100 Ruegeria mobilis NBRC 101030T (AB255401)

Fig. 1. Neighbour-joining phylogenetic tree generated by analysing nearly complete 16S rRNA gene sequences of strain Ad2T (in bold) and related Alphaproteobacteria. GenBank accession numbers are given in parentheses. The tree was constructed using maximum composite likelihood and pairwise deletion options. Bootstrap values (.50 % only) from 1000 resamplings are indicated at each node. Bar, 0.01 substitutions per nucleotide position.

Downloaded from www.microbiologyresearch.org by 142 International Journal of Systematic and Evolutionary Microbiology 63 IP: 54.70.40.11 On: Fri, 30 Nov 2018 17:58:27 Pseudovibrio axinellae sp. nov.

Physiological and biochemical tests were performed using Table 1. Differential features of strain Ad2T and Pseudovibrio API 20E, API 20NE and API ZYM kits (bioMe´rieux), as reference strains well as Biolog GN2 plates (Biolog). Tests were generally Strains: 1, Ad2T;2,P. denitrificans DSM 17465T;3,P. ascidiaceicola performed according to the manufacturer’s protocol except DSM 16392T;4,P. japonicus NCIMB 14279T. All data are from this that cells were resuspended in 3.33 % (w/v) artificial sea study, except where indicated, and all strains were tested concur- salts. All tests were performed in triplicate. API ZYM kits rently. +, Positive; 2, negative. were read after 5 h, API 20E and 20NE were read after 48 h, while Biolog GN2 plates were monitored for up to Characteristic 1 2 3 4 7 days. API 20NE data from assimilation tests were not used due to poor growth of the strains in API AUX Growth medium, despite supplementing it with 3 % (w/v) NaCl. Temperature range (uC) 10–30 18–37 15–30 15–35 Catalase activity was determined by gas production upon NaCl range (%) 1–4 2–4 2–4 2–5 exposure to 3 % (v/v) hydrogen peroxide. Cytochrome Enzyme activity + + oxidase activity was determined using Bactident Oxidase Tryptophan deaminase 2 2 +++ strips (Merck). Casein hydrolysis was determined on SYP- b-Galactosidase 2 a +++2 SW agar supplemented with 1 % (w/v) skimmed milk. -Glucosidase Acid production from: DNA hydrolysis was determined by growth on DNase test D-Galactose 2 ++2 agar (Fluka Biochemika) supplemented with NaCl to a Maltose +++2 final concentration of 3 % (w/v), followed by flooding the Sucrose +++2 agar plates with 1 M HCl. Gram staining, agar hydrolysis, Oxidation of: starch hydrolysis and methyl red tests were performed Tween 40 + 22+ according to the methods of Smibert & Kreig (1994). Acid N-Acetyl-D-galactosamine 22+ 2 production from a range of carbohydrates was determined N-Acetyl-D-glucosamine 22++ as reported by Leifson (1963). The ability to grow L-Fucose +++2 anaerobically was tested on SYP-SW agar at 24 uCinan D-Galactose 2 ++2 anaerobic jar. The test for denitrification was carried out as Gentiobiose 22++ described previously (Shieh et al., 2004). To test the myo-Inositol +++2 temperature range for growth, SYP-SW agar plates were Lactulose 2 ++2 +++ incubated at 4, 10, 15, 18, 24, 30, 35 and 37 uC. Growth at Maltose 2 ++ different pH was tested at 24 uC on SYP-SW agar at pH 5– Melibiose 2 2 + 9 buffered with 10 mM MES (pH 5–6), 10 mM HEPES Methyl b-D-glucoside 22 2 +++ (pH 7) or 10 mM Tris (pH 8–9). Salt tolerance was tested Raffinose 2 +++2 on SYP agar [1 % (w/v) starch, 0.4 % (w/v) yeast extract, Sucrose Turanose +++2 0.2 % (w/v) peptone, 1.5 % (w/v) agar] supplemented with Pyruvic acid methyl ester + 2 ++ u 0–10 % NaCl at 24 C. Plates were incubated for up to cis-Aconitic acid 22+ 2 7 days for all assays. Differential features between strain D 2 + 22 T -Galacturonic acid Ad2 and the reference strains of the three Pseudovibrio a-Ketobutyric acid + 2 + 2 species are listed in Table 1. Data from all phenotypic tests a-Ketoglutaric acid +++2 performed are listed in Table S2. Succinic acid + 2 + 2 + On the basis of phylogenetic analysis, DNA–DNA hybrid- Bromosuccinic acid 222 22++ ization and differential phenotypic characteristics, strain Succinamic acid D-Alanine + 2 ++ Ad2T is proposed as the type strain of a novel species, for L-Asparagine + 2 ++ which the name Pseudovibrio axinellae sp. nov. is proposed. Glycyl L-glutamic acid +++2 Hydroxy-L-proline +++2 Description of Pseudovibrio axinellae sp. nov. L-Threonine 22++ + ++ Pseudovibrio axinellae (a.xi.nel9la.e. N.L. gen. n. axinellae of Uridine 2 + + Axinella, the genus name of the marine sponge Axinella 2-Aminoethanol 2 2 D 22+ 2 dissimilis from which the type strain was isolated). -Glucose 6-phosphate DNA G+C content (mol%) 50.5 51.7* 51.2D 51.7d Cells are Gram-negative, rod-shaped, 2.0–3.5 mm long by 0.7–1.0 mm wide, and motile by means of subpolar flagella. *Data from Shieh et al. (2004). Colonies are circular, smooth and beige in colour with an DData from Fukunaga et al. (2006). entire margin on SYP-SW agar. Grows at 10–30 uC, pH 6– dData from Hosoya & Yokota (2007). 9 and 2–4 % (w/v) NaCl; grows weakly on 1 % (w/v) NaCl. Optimum growth conditions on SYP agar are at 24 uC, pH 7 and 3 % (w/v) NaCl. Catalase- and oxidase-positive. Grows anaerobically on SYP-SW agar. Nitrate is reduced to

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 143 IP: 54.70.40.11 On: Fri, 30 Nov 2018 17:58:27 J. A. O’Halloran and others nitrogen. Aesculin, casein, DNA and gelatin are hydrolysed, Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). while starch and agar are not. Acid is produced from D- A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466. glucose, maltose, D-mannose and sucrose, but not from cellobiose, D-fructose, D-galactose, a-lactose or D-xylose. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative Methyl red test is weakly positive. Can oxidize the measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142. following carbon sources in Biolog GN2 plates: dextrin, Enticknap, J. J., Kelly, M., Peraud, O. & Hill, R. T. (2006). Tween 40, Tween 80, L-fucose, a-D-glucose, myo-inositol, Characterization of a culturable alphaproteobacterial symbiont maltose, D-mannose, raffinose, sucrose, trehalose, turanose, common to many marine sponges and evidence for vertical pyruvic acid methyl ester, D-gluconic acid, D-glucuronic transmission via sponge larvae. Appl Environ Microbiol 72, 3724– acid, b-hydroxybutyric acid, a-ketobutyric acid, a-ketoglu- 3732. taric acid, DL-lactic acid, succinic acid, bromosuccinic acid, Felsenstein, J. (1985). Confidence limits on phylogenies: an approach D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L- using the bootstrap. Evolution 39, 783–791. aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl Fitch, W. M. (1971). Towards defining the course of evolution: L-glutamic acid, hydroxyl-L-proline, L-proline, L-serine, minimum change for a specific tree topology. Syst Zool 20, 406–416. inosine, uridine, thymidine, 2-aminoethanol and glycerol. Flemer, B., Kennedy, J., Margassery, L. M., Morrissey, J. P., O’Gara, F. Produces acetoin, but does not produce H2S or indole & Dobson, A. D. W. (2012). Diversity and antimicrobial activities of from tryptophan. Does not utilize citrate. Positive for microbes from two Irish marine sponges, Suberites carnosus and alkaline phosphatase, esterase (C4), leucine arylamidase Leucosolenia sp. J Appl Microbiol 112, 289–301. and a-glucosidase activity. Weakly positive for esterase Fukunaga, Y., Kurahashi, M., Tanaka, K., Yanagi, K., Yokota, A. & lipase (C8), valine arylamidase, trypsin, acid phosphatase Harayama, S. (2006). Pseudovibrio ascidiaceicola sp. nov., isolated and naphthol-AS-BI-phosphohydrolase activity. Negative from ascidians (sea squirts). Int J Syst Evol Microbiol 56, 343–347. for b-galactosidase, arginine dihydrolase, lysine decarboxy- Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert, C. & lase, ornithine decarboxylase, urease, tryptophan deami- Hacker, J. (2001). Isolation and phylogenetic analysis of bacteria with nase, lipase (C14), cystine arylamidase, a-chymotrypsin, antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microbiol Ecol 35, 305–312. a-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl- b-glucosaminidase, a-mannosidase and a-fucosidase activ- Hosoya, S. & Yokota, A. (2007). Pseudovibrio japonicus sp. nov., isolated from coastal seawater in Japan. Int J Syst Evol Microbiol 57, ity. The major fatty acid is 18 : 1v7c. 1952–1955. The type strain, Ad2T (5DSM 24994T5NCIMB 14761T), Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the was isolated from a marine sponge, Axinella dissimilis, spectrophotometric determination of DNA hybridization from collected in a marine lake (Lough Hyne) in Co. Cork, renaturation rates. Syst Appl Microbiol 4, 184–192. Ireland. The DNA G+C content of the type strain is Kennedy, J., Baker, P., Piper, C., Cotter, P. D., Walsh, M., Mooij, M. J., 50.5 mol%. Bourke, M. B., Rea, M. C., O’Connor, P. M. & other authors (2009). Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Acknowledgements Mar Biotechnol (NY) 11, 384–396. Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., This research was supported in part by grants awarded by the Jeon, Y. S., Lee, J.-H. & other authors (2012). Introducing EzTaxon- Department of Agriculture, Fisheries and Food (FIRM 08/RDC/629, e: a prokaryotic 16S rRNA gene sequence database with phylotypes DAF RSF 06 321, DAF RSF 06 377), the Irish Marine Institute that represent uncultured species. Int J Syst Evol Microbiol 62, 716– (C&CRA 2007/082) under the strategic Marine Biodiscovery RTDI 721. Programme and the Marine Biodiscovery Research award funded by Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty the Irish Government under the National Development Plan (2007- acids, antibiotic resistance, and deoxyribonucleic acid homology 2013), the Science Foundation of Ireland (07/IN.1/B948, 08/RFP/ groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361. GEN1295, SFI09/RFP/BMT2350), the Environmental Protection Agency (EPA 2006-PhD-S-21, EPA 2008-PhD-S-2), the Health Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acids Research Board (RP/2006/271, RP/2007/290, HRA/2009/146), Techniques in Bacterial Systematics, pp. 115–175. Edited by the Higher Education Authority (PRTLI4), the Irish Research E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Council for Science, Engineering and Technology (05/EDIV/FP107/ Leifson, E. (1963). Determination of carbohydrate metabolism of INTERPAM), and the European Commission (MTKD-CT2006- marine bacteria. J Bacteriol 85, 1183–1184. 042062, O36314, MicroB3-287589-OCEAN2012, MACUMBA-CP- Menezes, C. B., Bonugli-Santos, R. C., Miqueletto, P. B., Passarini, TP 311975, PharmaSea-CP-TP 312184). We would like to thank M. R., Silva, C. H., Justo, M. R., Leal, R. R., Fantinatti-Garboggini, F., Bernard Picton (Ulster Museum) and Dr Robert McAllen (UCC) for Oliveira, V. M. & other authors (2010). Microbial diversity associated their help in the collection and identification of sponge samples. with algae, ascidians and sponges from the north coast of Sa˜o Paulo state, Brazil. Microbiol Res 165, 466–482. References Mesbah, M., Premachandran, U. & Whitman, W. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high Altschul, S. F., Madden, T. L., Scha¨ ffer, A. A., Zhang, J., Zhang, Z., performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new Miller, L. T. (1982). Single derivatization method for routine analysis generation of protein database search programs. Nucleic Acids Res 25, of bacterial whole-cell fatty acid methyl esters, including hydroxy 3389–3402. acids. J Clin Microbiol 16, 584–586.

Downloaded from www.microbiologyresearch.org by 144 International Journal of Systematic and Evolutionary Microbiology 63 IP: 54.70.40.11 On: Fri, 30 Nov 2018 17:58:27 Pseudovibrio axinellae sp. nov.

Muscholl-Silberhorn, A., Thiel, V. & Imhoff, J. F. (2008). Abundance Smibert, R. M. & Kreig, N. R. (1994). Phenotypic characteristics. In and bioactivity of cultured sponge-associated bacteria from the Methods for General and Molecular Biology, pp. 607–654. Edited by Mediterranean sea. Microb Ecol 55, 94–106. P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. O’Halloran, J. A., Barbosa, T. M., Morrissey, J. P., Kennedy, J., O’Gara, Washington, DC: American Society for Microbiology. F. & Dobson, A. D. W. (2011). Diversity and antimicrobial activity of Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular Pseudovibrio spp. from Irish marine sponges. J Appl Microbiol 110, evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol 1495–1508. Evol 24, 1596–1599. Penesyan, A., Tebben, J., Lee, M., Thomas, T., Kjelleberg, S., Harder, Thiel, V. & Imhoff, J. F. (2003). Phylogenetic identification of bacteria T. & Egan, S. (2011). Identification of the antibacterial compound with antimicrobial activities isolated from Mediterranean sponges. produced by the marine epiphytic bacterium Pseudovibrio sp. D323 Biomol Eng 20, 421–423. and related sponge-associated bacteria. Mar Drugs 9, 1391–1402. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. strategies for multiple sequence alignment aided by quality analysis Santos, O. C., Pontes, P. V., Santos, J. F., Muricy, G., Giambiagi- tools. Nucleic Acids Res 25, 4876–4882. deMarval, M. & Laport, M. S. (2010). Isolation, characterization and Vizcaino, M. I. (2011). The chemical defense of Pseudopterogorgia phylogeny of sponge-associated bacteria with antimicrobial activities americana: a focus on the antimicrobial potential of a Pseudovibrio sp. from Brazil. Res Microbiol 161, 604–612. PhD thesis, University of South Carolina, USA. Sertan-de Guzman, A. A., Predicala, R. Z., Bernardo, E. B., Neilan, Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, B. A., Elardo, S. P., Mangalindan, G. C., Tasdemir, D., Ireland, C. M., O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & Barraquio, W. L. & Concepcion, G. P. (2007). Pseudovibrio denitrificans other authors (1987). International Committee on Systematic strain Z143-1, a heptylprodigiosin-producing bacterium isolated from Bacteriology. Report of the ad hoc committee on reconciliation of a Philippine tunicate. FEMS Microbiol Lett 277, 188–196. approaches to bacterial systematics. Int J Syst Bacteriol 37, 463– Shieh, W. Y., Lin, Y.-T. & Jean, W. D. (2004). Pseudovibrio denitrificans 464. gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative Webster, N. S. & Hill, R. T. (2001). The culturable microbial bacterium capable of denitrification. Int J Syst Evol Microbiol 54, community of the Great Barrier Reef sponge Rhopaloeides odorabile 2307–2312. is dominated by an a-proteobacterium. Mar Biol 138, 843–851.

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 145 IP: 54.70.40.11 On: Fri, 30 Nov 2018 17:58:27