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

A Gram-negative, motile, rod-shaped bacterial strain, designated Ad2, was isolated from a marine sponge, Axinella dissimilis, which was collected from a semi-enclosed marine lake in Ireland. Strain Ad2 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 Ad2 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 Ad2 and the type strains of other Pseudovibrio species were ,27 %. The DNA G+C content of strain Ad2 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 Ad2 represented a novel species of the genus Pseudovibrio. The name Pseudovibrio axinellae sp. nov. is proposed, with Ad2 (5DSM 249945NCIMB 14761) as the type strain.

The genus Pseudovibrio was first described by Shieh et al. (2004) and, at the time of writing, comprised three species: Pseudovibrio denitrificans (Shieh et al., 2004) and Pseudovibrio japonicus (Hosoya & Yokota, 2007), whose type strains were isolated from seawater, and Pseudovibrio ascidiaceicola (Fukunaga et al., 2006), whose type strain was isolated from a sea squirt (ascidian).In the search for clinically relevant antimicrobial compounds, members of the genus Pseudovibrio are of interest due to their bioactive potential (Kennedy et al., 2009;Santos et al., 2010;O'Halloran et al., 2011;Flemer et al., 2012), and a number of antimicrobial compounds from members of the genus Pseudovibrio have been characterized (Sertan-de Guzman et al., 2007;Penesyan et al., 2011;Vizcaino, 2011).Members of the genus Pseudovibrio are frequently isolated from sponges (Hentschel et al., 2001;Webster & Hill, 2001;Thiel & Imhoff, 2003;Enticknap et al., 2006;Muscholl-Silberhorn et al., 2008;Kennedy et al., 2009;Menezes et al., 2010;Santos et al., 2010) and we have previously analysed a population of Pseudovibrio species isolated from a number of marine sponges (O'Halloran et al., 2011).One of the isolates, Ad2 T , which displayed narrow-spectrum antimicrobial activity, appeared to represent a distinct species based on 16S rRNA gene sequencing and randomly amplified polymorphic DNA analysis.Here, we report on the characterization of strain Ad2 T using a polyphasic approach.
Colonies of strain Ad2 T were circular, smooth and beige in colour with an entire margin on SYP-SW agar.Phasecontrast microscopy of early stationary phase cells of strain Ad2 T grown in SYP-SW broth at 24 u C revealed that the cells were rod-shaped and motile, 2.0-3.5 mm long by 0.7-1.0mm wide.Transmission electron microscopy of cells of strain Ad2 T negatively stained with uranyl acetate showed that, similarly to the previously reported type strains of species of the genus Pseudovibrio, strain Ad2 T cells possessed one to several subpolar flagella (Fig. S1, available in IJSEM Online).
For phylogenetic analysis, DNA was extracted from strain Ad2 T using an UltraClean microbial DNA isolation kit The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Ad2 T is JN167515.(MoBio) according to the manufacturer's protocol.To amplify the 16S rRNA gene, the universal primers 27f (59-AGAGTTTGATCMTGGCTCAG-39) and 1492r (59-TACG-GYTACCTTGTTACGACTT-39) were used (Lane, 1991) with a thermal cycling programme of 95 u C for 5 min, followed by 30 cycles of 95 u C for 30 s, 50 u C for 30 s and 72 u C for 90 s, and a final elongation step of 72 u C for 7 min.The amplification product was purified using a QIAquick PCR purification kit (Qiagen), cloned using a TOPO TA cloning kit (Invitrogen), and sequenced by GATC Biotech using the M13 forward (59-GTAAA-ACGACGGCCAG-39) and M13 reverse (59-CAGGAAA-CAGCTATGAC-39) primers.The nearly complete (1446 bp) sequence of the 16S rRNA gene of strain Ad2 T was compared with sequences in the GenBank database using BLAST (Altschul et al., 1997) and aligned with reference 16S rRNA gene sequences using CLUSTAL X (Thompson et al., 1997).Phylogenetic analyses were carried out using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods using MEGA4 (Tamura et al., 2007).Bootstrap tests (Felsenstein, 1985) were performed 1000 times.The neighbour-joining phylogenetic analysis indicated that strain Ad2 T was a member of the genus Pseudovibrio (Fig. 1), and this was supported by the maximumparsimony phylogenetic analysis, where a similar tree topology was observed (Fig. S2).The levels of 16S rRNA gene sequence similarity between strain Ad2 T and P. ascidiaceicola F423 T , P. denitrificans DN34 T and P. japonicus WSF2 T were calculated using the EzTaxon-e tool (Kim et al., 2012) as 98.2, 97.3 and 98.3 %, respectively.DNA-DNA hybridization and determination of the DNA G+C content of strain Ad2 T were carried out by the DSMZ, Braunschweig, Germany, where cells were disrupted using a French press and the DNA in the crude lysate was purified according to the method of Cashion et al. (1977).DNA-DNA hybridization was performed in duplicate and carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983).Strain Ad2 T showed low DNA-DNA hybridization values to P. ascidiaceicola DSM 16392 T , P. denitrificans DSM 17465 T , and P. japonicus NCIMB 14279 T , with average values of 26.1, 18.1 and 15.7 %, respectively.These values are far below the recommended threshold value of 70 % for delineation of bacterial species (Wayne et al., 1987).The DNA G+C content of strain Ad2 T was 50.5 mol% according to the method of Mesbah et al. (1989).
The cellular fatty acid contents of strain Ad2 T and Pseudovibrio reference strains were analysed using the Microbial Identification system (MIDI) and carried out by the DSMZ, where fatty acid methyl esters were obtained from 40 mg of cells grown for 24 h at 28 u C on marine agar (Difco) by saponification, methylation and extraction according to the methods of Miller (1982) and Kuykendall et al. (1988).Similarly to virtually all members of the Alphaproteobacteria, the major fatty acid of strain Ad2 T was 18 : 1v7c.In contrast to the three Pseudovibrio reference strains, 16 : 0 and summed feature 3 (15 : 0 iso 2-OH and/or 16 : 1v7c) also represented a significant proportion (.10 %) of the cellular fatty acids in strain Ad2 T , while 15 : 0 and 17 : 0 were also detected in strain Ad2 T but not in the reference strains.Detailed information on the cellular fatty acid composition of strain Ad2 T and Pseudovibrio reference strains is provided in Table S1.Physiological and biochemical tests were performed using API 20E, API 20NE and API ZYM kits (bioMe ´rieux), as well as Biolog GN2 plates (Biolog).Tests were generally performed according to the manufacturer's protocol except that cells were resuspended in 3.33 % (w/v) artificial sea salts.All tests were performed in triplicate.API ZYM kits were read after 5 h, API 20E and 20NE were read after 48 h, while Biolog GN2 plates were monitored for up to 7 days.API 20NE data from assimilation tests were not used due to poor growth of the strains in API AUX medium, despite supplementing it with 3 % (w/v) NaCl.Catalase activity was determined by gas production upon exposure to 3 % (v/v) hydrogen peroxide.Cytochrome oxidase activity was determined using Bactident Oxidase strips (Merck).Casein hydrolysis was determined on SYP-SW agar supplemented with 1 % (w/v) skimmed milk.DNA hydrolysis was determined by growth on DNase test agar (Fluka Biochemika) supplemented with NaCl to a final concentration of 3 % (w/v), followed by flooding the agar plates with 1 M HCl.Gram staining, agar hydrolysis, starch hydrolysis and methyl red tests were performed according to the methods of Smibert & Kreig (1994).Acid production from a range of carbohydrates was determined as reported by Leifson (1963).The ability to grow anaerobically was tested on SYP-SW agar at 24 u C in an anaerobic jar.The test for denitrification was carried out as described previously (Shieh et al., 2004).To test the temperature range for growth, SYP-SW agar plates were incubated at 4, 10, 15, 18, 24, 30, 35 and 37 u C. Growth at different pH was tested at 24 u C on SYP-SW agar at pH 5-9 buffered with 10 mM MES (pH 5-6), 10 mM HEPES (pH 7) or 10 mM Tris (pH 8-9).Salt tolerance was tested on SYP agar [1 % (w/v) starch, 0.4 % (w/v) yeast extract, 0.2 % (w/v) peptone, 1.5 % (w/v) agar] supplemented with 0-10 % NaCl at 24 u C. Plates were incubated for up to 7 days for all assays.Differential features between strain Ad2 T and the reference strains of the three Pseudovibrio species are listed in Table 1.Data from all phenotypic tests performed are listed in Table S2.
On the basis of phylogenetic analysis, DNA-DNA hybridization and differential phenotypic characteristics, strain Ad2 T is proposed as the type strain of a novel species, for which the name Pseudovibrio axinellae sp.nov. is proposed.
Pseudovibrio axinellae (a.xi.nel9la.e.N.L. gen.n. axinellae of Axinella, the genus name of the marine sponge Axinella dissimilis from which the type strain was isolated).
Cells are Gram-negative, rod-shaped, 2.0-3.5 mm long by 0.7-1.0mm wide, and motile by means of subpolar flagella.Colonies are circular, smooth and beige in colour with an entire margin on SYP-SW agar.Grows at 10-30 u C, pH 6-9 and 2-4 % (w/v) NaCl; grows weakly on 1 % (w/v) NaCl.Optimum growth conditions on SYP agar are at 24 u C, pH 7 and 3 % (w/v) NaCl.Catalase-and oxidase-positive.Grows anaerobically on SYP-SW agar.Nitrate is reduced to nitrogen.Aesculin, casein, DNA and gelatin are hydrolysed, while starch and agar are not.Acid is produced from D- glucose, maltose, D-mannose and sucrose, but not from cellobiose, D-fructose, D-galactose, a-lactose or D-xylose.
The type strain, Ad2 T (5DSM 24994 T 5NCIMB 14761 T ), was isolated from a marine sponge, Axinella dissimilis, collected in a marine lake (Lough Hyne) in Co. Cork, Ireland.The DNA G+C content of the type strain is 50.5 mol%.

Fig. 1 .
Fig.1.Neighbour-joining phylogenetic tree generated by analysing nearly complete 16S rRNA gene sequences of strain Ad2 T (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.
Two supplementary tables and two supplementary figures are available with the online version of this paper.