Salipiger mucescens gen. nov., sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium isolated from hypersaline soil, belonging to the {alpha}-Proteobacteria

Salipiger mucescens gen. nov., sp. nov. is a moderately halophilic, exopolysaccharide-producing, Gram-negative rod isolated from a hypersaline habitat in Murcia in south-eastern Spain. The bacterium is chemoheterotrophic and strictly aerobic (i.e. unable to grow under anaerobic conditions either by fermentation or by nitrate or fumarate respiration). It does not synthesize bacteriochlorophyll a. Catalase and phosphatase are positive. It does not produce acids from carbohydrates. It cannot grow with carbohydrates or amino acids as sole sources of carbon and energy. It grows best at 910 % w/v NaCl and requires the presence of Na⁺ but not Mg²⁺ or K⁺, although they do stimulate its growth somewhat when present. Its major fatty-acid component is 18 : 1ω7c (78·0 %). The predominant respiratory lipoquinone found in strain A3^T is ubiquinone with ten isoprene units. The G+C content is 64·5 mol%. Phylogenetic analyses strongly indicate that this strain forms a distinct line within a clade containing the genus Roseivivax in the subclass α-Proteobacteria. The similarity value with Roseivivax halodurans and Roseivivax halotolerans is 94 %. In the light of the polyphasic evidence gathered in this study it is proposed that the isolate be classified as representing a new genus and species, Salipiger mucescens gen. nov., sp. nov. The proposed type strain is strain A3^T (=CECT 5855^T=LMG 22090^T=DSM 16094^T).

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Abbreviations: EPS, exopolysaccharide; PHA, poly-β-hydroxyalkanoate

Published online ahead of print on 9 July 2004 as DOI 10.1099/ijs.0.63166-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A3^T is AY527274.

Moderately halophilic bacteria are widely distributed throughout hypersaline habitats and require from 3 to 15 % w/v NaCl for satisfactory growth (Kushner & Kamekura, 1988). In recent years it has been found that several products of these bacteria, such as exopolysaccharides (EPSs), halophilic enzymes and compatible solutes, may have very useful applications in biotechnology (Ventosa, 2004; Ventosa et al., 1998). During an extensive search of many different hypersaline habitats in Spain and Morocco designed to obtain new EPSs we discovered that the commonest halophilic EPS producers were various novel species of the genus Halomonas, most importantly Halomonas maura and Halomonas eurihalina (Bouchotroch et al., 2001; Martínez-Cánovas et al., 2004c; Quesada et al., 1990, 2004), together, to a lesser extent, with Halomonas ventosae (Martínez-Cánovas et al., 2004a) and Halomonas anticariensis (Martínez-Cánovas et al., 2004b). We describe and classify here a hitherto unassigned halophilic EPS-producing strain that was also isolated in these studies. On the basis of its phenotypic features, comparative studies of its 16S rRNA gene sequence and investigations into its polar-lipid and isoprenoid quinone contents, together with its salt requirements and its inability to synthesize bacteriochlorophyll a, we propose that this bacterium should be assigned to a new genus, Salipiger, with a single species Salipiger mucescens gen. nov., sp. nov.

The strain named A3^T was isolated from a saline soil bordering a saltern on the Mediterranean coast at Calblanque (Murcia, south-eastern Spain) (Martínez-Cánovas et al., 2004c). The strain was routinely grown at 32 °C in MY medium (Quesada et al., 1993) supplemented with a 7·5 % w/v sea-salt solution (Rodríguez-Valera et al., 1981).

An initial phenotypic study including 135 tests was undertaken by Martínez-Cánovas et al. (2004c). Salt requirements and optimum salt concentration were determined in MY medium according to the methods described by Bouchotroch et al. (2001). The salt concentrations assayed ranged from 0·5 to 30 % w/v and were prepared from a mixture of sea salts according to Rodríguez-Valera et al. (1981). We also tested to see whether strain A3^T could survive with NaCl alone or whether it required other magnesium and/or potassium salts.

Bacteriochlorophyll a was analysed spectrophotometrically using the procedure of Cohen-Bazire et al. (1957) following the recommendations of Allgaier et al. (2003). Two microlitres of a liquid culture of strain A3^T incubated in the dark was centrifuged and the pellet resuspended in a drop of the remaining medium. A 1·5 ml volume of an ice-cold (20 °C) acetone/methanol solution (7 : 2 v/v) was added, mixed thoroughly and incubated at room temperature in the dark for 12 h. After centrifugation, spectrophotometric measurements were made at 600900 nm.

Fatty acids and quinones were identified by high-resolution GLC and HPLC respectively at the DSMZ.

Transmission electron micrographs were made using the methods described by Bouchotroch et al. (2001).

The 16S rRNA gene was amplified by PCR using standard protocols (Saiki et al., 1988). The forward primer, 16F27 (5'-AGAGTTTGATCMTGGCTCAG-3'), annealed at positions 827 and the reverse primer, 16R1488 (5'-CGGTTACCTTGTTAGGACTTCACC-3') (both from Pharmacia), annealed at the complement of positions 15111488 (Escherichia coli numbering according to Brosius et al., 1978). The PCR products were purified using the QIAquick spin-gel extraction kit (Qiagen). Direct sequence determinations of PCR-amplified DNAs were carried out with the ABI PRISM dye-terminator cycle-sequencing ready-reaction kit (Perkin-Elmer) and an ABI PRISM 377 sequencer (Perkin-Elmer) according to the manufacturer's instructions. The sequences obtained were compared to reference 16S rRNA gene sequences available in the GenBank, EMBL and DDBJ databases obtained from the National Center of Biotechnology Information database using the BLAST search. Phylogenetic analysis was performed using the software MEGA version 2.1 (Kumar et al., 2001) after multiple alignments of data by CLUSTAL X (Thompson et al., 1997). Distances and clustering were determined using the neighbour-joining and maximum-parsimony methods. The stability of clusters was ascertained by performing a bootstrap analysis (1000 replications).

The strain described here was isolated during a wide research programme, the main objective of which was to identify EPS-producing bacteria in different hypersaline habitats (Martínez-Cánovas et al., 2004c; Quesada et al., 2004). Numerical analysis of its phenotypic characteristics demonstrated that strain A3^T was not related to other halophilic EPS-producing strains isolated from these habitats (Martínez-Cánovas et al., 2004c). A3^T was also found to contain at least seven plasmids (550, 467, 184, 140·8, 110·6, 98·2 and 30·8 kb). It did not however contain the 600 kb megaplasmid found in other halophilic micro-organisms (Argandoña et al., 2003). All these facts led us to characterize this strain further and eventually to propose its assignment to a new genus.

Strain A3^T is strictly halophilic, being unable to grow in the absence of sea salts. It is an aerobic chemo-organotroph, unable to grow under anaerobic conditions either by fermenting sugars or by reducing nitrate, nitrite or fumarate. Bacteriochlorophyll a was not detected. It is characterized by its low nutritional and biochemical versatility. Its phenotypic characteristics appear in the species description. Phenotypic features that differentiate strain A3^T from the two species of Roseivivax and other members of the family Rhodobacteraceae related to it phylogenetically are shown in Table 1. The data included in this table demonstrate that there is no phenotypic similarity between A3^T and the other strains concerned. The G+C content of strain A3^T is 64·5 mol% (Martínez-Cánovas et al., 2004c), which is similar to the value of 64·4 mol% obtained for Roseivivax halodurans (Suzuki et al., 1999).

Table 1. Characteristics that distinguish Salipiger mucescens gen. nov., sp. nov. A3T from other related members of the family Rhodobacteraceae Reference strains: 1 and 2, Roseivivax halodurans JCM 10272T and Roseivivax halotolerans JCM 10271T (Suzuki et al., 1999; Nishimura et al., 1994); 3, Antarctobacter heliothermus DSM 11445T (Labrenz et al., 1998); 4, Ketogulonicigenium vulgare DSM 405T (Urbance et al., 2001); 5, Jannaschia helgolandensis DSM 14858T (Wagner-Döbler et al., 2003); 6, Leisingera methylohalidivorans ATCC BAA-92T (Schaefer et al., 2002); 7, Octadecabacter arcticus CIP 106731T (Gosink et al., 1997); 8, Roseobacter litoralis (Shiba, 1991; Labrenz et al., 1998); 9, Roseovarius tolerans DSM 11457T (Labrenz et al., 1999); 10, Ruegeria algicola ATCC 51440T (Lafay et al., 1995; Labrenz et al., 1998); 11, Sagittula stellata ATCC 700073T (González et al., 1997); 12, Silicibacter lacuscaerulensis DSM 11314T (Petursdottir & Kristjansson, 1997); 13, Sulfitobacter mediterraneus DSM 12244T (Pukall et al., 1999); 14, Staleya guttiformis DSM 11443T (Labrenz et al., 2000). +, Positive; , negative; W, weak; ND, no data available.

Strain A3^T contains a large quantity (78·0 %) of cis-11 octadecenoic acid (18 : 1ω7c) in combination with 16 : 0, 18 : 0, 11-methyl-branched cis-9 octadecenoic acid (11-methyl 18 : 1ω7c) and 3-hydroxy 12 : 0 (12·4, 2·0, 1·9 and 2·3 %, respectively). The presence of 18 : 1ω7c as the predominant fatty acid is a feature characteristic of several major taxa within the α-Proteobacteria (Table 1). Nevertheless, strain A3^T also contains cyclo-substituted fatty acids (2·3 %), which are not widely present in the family Rhodobacteraceae. The fatty acids of the Roseivivax species, which are most phylogenetically related to A3^T, have not been thoroughly described, although in a study published before the taxonomical description of Roseivivax halodurans and Roseivivax halotolerans Nishimura et al. (1994) reported that the main cellular fatty-acid component in these bacteria was 18 : 1. The only respiratory lipoquinone detected was ubiquinone 10. The presence of ubiquinone 10 as the dominant respiratory lipoquinone is characteristic of members of the α-Proteobacteria.

Fig. 1 shows the cell morphology of strain A3^T. Thin sections reveal a typical Gram-negative cell-envelope profile; the cell contains poly-β-hydroxyalkanoate (PHA) granules. EPS appears associated with the cell surface.

(145K): Fig. 1. Transmission electron micrograph of cells of strain A3T stained with ruthenium red. Bar, 1 µm.

According to the recommendations of Stackebrandt et al. (2002) we determined the almost complete 16S rRNA gene sequence of strain A3^T (1364 bp), corresponding to positions 461445 of the Escherichia coli 16S rRNA gene. The phylogenetic tree obtained via the neighbour-joining method is shown in Fig. 2. The maximum-parsimony algorithm gave a similar result (data not shown). Together with the sequence of A3^T, our phylogenetic analysis also included all the representatives of the family Rhodobacteraceae described to date plus four halophilic bacteria belonging to the γ-Proteobacteria as an outgroup. Strain A3^T belongs to a clade containing Roseivivax halodurans and Roseivivax halotolerans and shows 94 % similarity to both species. This value indicates the possibility of a new taxon of at least genus status. The physiology of strain A3^T is also clearly different from that of the Roseivivax species, which are pink-pigmented chemoheterotrophs that synthesize bacteriochlorophyll a under aerobic conditions, produce acids from sugars and are able to use different compounds as sole sources of carbon and energy. Thus, on the basis of phylogenetic evidence, differences in phenotypic characteristics and its inability to synthesize bacteriochlorophyll a, we are of the opinion that strain A3^T should be recognized as a new genus with a single species, for which we propose the name Salipiger mucescens gen. nov., sp. nov. The genus is in the same clade as that of Roseivivax, belonging to the α-3 group of the α-subclass of the Proteobacteria, within the family Rhodobacteraceae (Garrity & Holt, 2001). Members of this taxon are in general non-sulfur, purple bacteria that carry out anoxygenic photosynthesis. This family includes a group of aerobic bacteriochlorophyll-containing bacteria (ABC), to which Roseivivax pertains (Imhoff & Madigan, 2002). Roseivivax is taxonomically related to the Roseobacter clade, a group of 11 genera of the family Rhodobacteraceae (Allgaier et al., 2003), which make up the most abundant populations in marine habitats (González & Moran, 1997).

(46K): Fig. 2. Phylogenetic relationships amongst Salipiger mucescens A3T and members of other genera of the family Rhodobacteraceae plus other taxa of Gram-negative halophilic bacteria. The tree was constructed using the neighbour-joining algorithm. Only bootstrap values above 50 % are shown (1000 replications). Bar, 2 % estimated sequence divergence.

Description of Salipiger gen. nov.
Salipiger (Sa.li.pi'ger. L. masc. sb. sal salt; L. masc. adj. piger lazy; N.L. masc. sb. Salipiger lazy halophile).

Gram-negative, non-motile rods, 22·5 µm long by 0·751 µm wide. Chemoheterotrophic, strictly aerobic, being unable to grow under anaerobic conditions either by fermentation, nitrate or fumarate reduction or photoheterotrophy. PHA, cytochrome oxidase and catalase are present. Low nutritional and biochemical versatility. Strictly halophilic, requiring Na⁺ ions for growth. The principal cellular fatty acids are 18 : 1ω7c and 16 : 0. It has ubiquinone with ten isoprene units. The type species is Salipiger mucescens.

Description of Salipiger mucescens sp. nov.
Salipiger mucescens (mu.ces'cens. L. masc. ppl. adj. mucescens slimy).

In addition to the traits reported for the genus, the species grows on MY solid medium in the form of circular, convex, cream-coloured, mucoid colonies. In liquid medium its growth pattern is uniform. The cells are encapsulated. It is moderately halophilic, capable of growing in salt concentrations (mixture of sea salts) from 0·5 to 20 % w/v, the optimum range being between 3 and 6 % w/v. Nevertheless, if NaCl is substituted for sea salts, the concentration required for optimum growth reaches values of 9 to 10 % w/v. It grows within the temperature range of 20 to 40 °C and at pH values of between 6 and 10. It produces H₂S from L-cysteine. Selenite reduction, gluconate oxidation and phosphatase are positive. Urea and Tween 20 are hydrolysed. It does not produce acids from the following sugars: adonitol, D-cellobiose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, maltose, D-mannitol, D-mannose, D-melezitose, L-rhamnose, sucrose, D-salicin, D-sorbitol, sorbose and D-trehalose. O/F, ONPG, indole, methyl-red and VogesProskauer are negative. Phenylalanine deaminase is not produced. Tyrosine, Tween 80, starch, aesculin, gelatin, DNA, lecithin and casein are not hydrolysed. Growth on either MacConkey or cetrimide agar is unviable. Blood is not haemolysed. Neither nitrate nor nitrite is reduced. The following compounds are not acceptable as sole carbon and energy sources: L-arabinose, D-cellobiose, aesculin, D-fructose, D-glucose, D-galactose, lactose, maltose, D-mannose, D-salicin, acetate, citrate, formate, fumarate, glycerol, lactate, malonate, propionate and succinate. The following compounds are not used as sole carbon, nitrogen and energy sources: L-alanine, L-cysteine, L-histidine, isoleucine, L-lysine, L-methionine, L-serine and L-valine. It is susceptible to (µg) amoxicillin (25), ampicillin (10), carbenicillin (100), cefotaxime (30), cefoxitin (30), chloramphenicol (30), erythromycin (15), kanamycin (30), nitrofurantoin (300), rifampicin (30), sulfonamide (250), tobramycin (10) and trimetoprim/sulphametoxazol (1·25/23·7), and is resistant to nalidixic acid (30) and polymixin B (300). The major fatty acids (%) are 18 : 1ω7c (78·0), 16 : 0 (12·4), 12 : 1 3-OH (2·3), 19 : 0 cyclo ω8c (2·3), 18 : 0 (2·0) and 16 : 1ω7c/15 : 0 iso 2-OH (1·3). Its DNA G+C content is 64·5 mol% (T_m method).

The type strain is strain A3^T (=CECT 5855^T=LMG 22090^T=DSM 16094^T), isolated from a hypersaline soil taken from a solar saltern in Calblanche (Murcia, south-eastern Spain).