International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1625–1632 Printed in Great Britain

Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium

Microbial Samir Bouchotroch, Emilia Quesada, Ana del Moral, Inmaculada Llamas Exopolysaccharides Research Group, Department of and Victoria Be! jar Microbiology, Faculty of Pharmacy, Campus Universitario de Cartuja, Author for correspondence: Emilia Quesada. Tel: j34 958 243871. Fax: j34 958 246235. University of Granada, e-mail: equesada!platon.ugr.es 18071 Granada, Spain

Four moderately halophilic, exopolysaccharide-producing bacterial strains isolated from soil samples collected from a saltern at Asilah (Morocco) are reported. These four strains were initially considered to belong to the genus . Their DNA GMC contents varied between 622 and 641 mol%. DNA–DNA hybridization revealed a considerable degree of DNA–DNA similarity amongst all four strains (755–808%). Nevertheless, similarity with the reference strains of phylogenetically close relatives was lower than 40%. 16S rRNA gene sequences were compared with those of other species of Halomonas and other Gram-negative and they were sufficiently distinct phylogenetically from other recognized Halomonas species to warrant their designation as a novel species. The name Halomonas maura sp. nov. is therefore proposed, with strain S-31T (l CECT 5298T l DSM 13445T) as the type strain. The fatty acid composition of strain S-31T revealed the presence of 18:1ω7c,16:1ω7c/2-OH i15:0 and 16:0 as the major components. Growth rate analysis showed that strain S-31T had specific cationic requirements for NaM and Mg2M.

Keywords: exopolysaccharides, moderately halophilic bacteria, Halomonas

INTRODUCTION In the course of our studies into hypersaline environ- ments, a group of Halomonas eurihalina strains were The family includes the genera isolated that synthesized large quantities of exopoly- Halomonas, Chromohalobacter and . These saccharides (EPS) (Be! jar et al., 1998; Quesada et al., genera form a group within the γ- and 1993). These polymers have since been characterized their 16S rRNA sequences contain common signature and evaluated for their potential applications in features (Dobson & Franzmann, 1996; Franzmann et industry (Calvo et al., 1995, 1998; Martı!nez-Checa et al., 1989). The genus Halomonas comprises slightly or al., 1996). EPS polymers are very useful in various moderately halophilic, chemo-organotrophic, Gram- fields of biotechnology, particularly in the food, negative rods, species of which are widely distributed pharmaceutical and petroleum industries (Sutherland, in hypersaline habitats. During the last decade, a large 1990, 1998; Tombs & Harding, 1998). Although the number of novel species has been assigned to the genus market for polymers already has recourse to gums of Halomonas, including some which had been originally remarkable quality, the diversity of micro-organisms assigned to the genera Deleya and Volcaniella (Mellado being discovered offers excellent prospects of finding et al., 1995; Quesada et al., 1984, 1990; Valderrama et new EPS with new or better properties. The polymers al., 1991; Ventosa et al., 1998), genera which have found in our laboratory, for example, possess ad- since been included within the genus Halomonas. vantageous qualities not shared by many poly- saccharides currently in use: they have simultaneous

...... emulsifying and viscosifying activities; some are Abbreviations: DIG, digoxigenin; EPS, exopolysaccharide; PHA, poly-β- able to gel at acidic pH; they are quite resistant to hydroxyalkanoate; Tm, midpoint value of the thermal denaturation profile. high osmotic strength; and are also thermostable The EMBL accession number for the 16S rDNA sequence of strain S-31T is (Bouchotroch et al., 2000; Calvo et al., 1998). We are AJ271864. carrying out a wide search aimed at isolating and

01591 # 2001 IUMS 1625 S. Bouchotroch and others characterizing EPS-producing, halophilic micro- DNA–DNA hybridization. Reference DNA was double- organisms. labelled by nick-translation using digoxigenin-11-2h-dUTP (DIG-11-dUTP) and biotin-16-dUTP (Boehringer Mann- Recently, the results of a taxonomic study of a group heim). The optimum ratio for the nucleotide mixture of 46 moderately halophilic, EPS-producing bacteria of DIG-11-dUTP:biotin-16-dUTP was 0n75:0n25 (v\v). isolated from hypersaline environments in Morocco DNA (0n5 µg) was labelled according to the protocol of the were reported. Their phenotypic features, which were manufacturer and resuspended in 100 µl distilled water. One subject to numerical analysis, were studied. Represen- microlitre was used to measure the efficiency of the labelling tative strains from two phena obtained (6 Gram- reaction. positive and 32 Gram-negative strains) were chosen to DNA–DNA hybridization was conducted following the determine their DNA base composition and the methods of Lind & Ursing (1986) with the modifications of percentage of DNA–DNA similarity to reference Ziemke et al. (1998). Between 40 and 60 ng labelled sheared strains. All indications were that the Gram-negative DNA was mixed with 15 µg unlabelled DNA in 0n1ml bacteria could be accommodated within the genus 0n28 M phosphate buffer (PB), pH 6n8. This mixture was Halomonas, but they could not be assigned to any of its denatured at 100 mC for 10 min and subsequently cooled in a recognized species (Bouchotroch et al., 1999). bath of iced water. The mixture was then incubated for 16 h at 30 mC below the Tm of the homologous DNA. Single- and The object of this present work has been to classify double-stranded DNA were eluted through hydroxyapatite accurately four strains of Halomonas isolated during (Brenner et al., 1969). Rehydrated hydroxyapatite (DNA our previous study. To this end, DNA–DNA simi- grade Bio-Gel HTP; Bio-Rad) was equilibrated with 0n14 M larities with other closely related species of Halomonas PB. The preparation was mixed thoroughly and incubated for 15 min at 5 mC below the temperature used in the were compared. The 16S rRNA gene sequences of the reassociation. The single- and the double-stranded DNA novel isolates were determined and compared to those were then eluted using four washes of 0n25 ml 0n14 M PB, of related taxa. Their polar lipids, growth rates under 0n2% (w\v) SDS and two washes of 0n2ml0n4 M PB. different saline conditions and cell morphology of strain S-31T, which was selected as the type strain of To detect the eluted DNA, 200 µl was incubated for 2 h on streptavidin-coated microtitre plates (Boehringer the newly proposed species, Halomonas maura sp. Mannheim) with 0n1% (w\v) BSA (Sigma). The double- nov., were also studied. stranded DNA was denatured and chilled on ice before incubation. Washes with PBS (0n3 M NaCl, 5 mM METHODS Na#HPO%,1n5mM KH#PO%,pH7n2) were followed by incubation for 1 h with anti-DIG antibodies conjugated Bacterial strains. The moderate used in this study, with alkaline phosphatase (Boehringer Mannheim) in 200 µl strains S-7, S-30, S-31T and S-36, were isolated from soil PBS and 0n1% (w\v) BSA. Both incubations were at room samples collected from a solar saltern at Asilah (Morocco). temperature with agitation. The wells were then washed with These four strains were initially identified as Halomonas spp. PBS. Detection was performed with 250 µl p-nitrophenyl (Bouchotroch et al., 1999). phosphate at 37 mC without agitation. Colour development was measured at 405 nm (Tilssen, 1985). Culture conditions. Unless otherwise stated, the strains were grown in MY medium (Quesada et al., 1993) with the The degree of reassociation (binding ratio) was expressed as −" following composition (g l ): NaCl, 51n3; MgCl#.6H#O, 9; the percentage of labelled DNA released with 0n4M PB MgSO%.7H#O, 13; CaCl#.2H#O, 0n2; KCl, 1n3; NaHCO$, compared to the total labelled DNA released. The relative 0n05; NaBr, 0n15; FeCl$.6H#O; trace metal solution, 0n00325; binding ratio of heterologous DNA was expressed as the glucose, 10; yeast extract, 3; malt extract, 3; proteose- percentage of homologous binding. Pooled SD of all " peptone, 5 (pH 7). Bacto agar (2 g l− ) was added for the experiments ranged from 1 to 4%. preparation of solid media. The strains were routinely grown PCR amplification of 16S rRNA genes. The 16S rRNA gene with shaking (orbital shaker, 100 r.p.m.) at 32 C in 500 ml m was amplified by PCR using the standard protocols (Saiki et Erlenmeyer flasks containing 100 ml medium. al., 1988). The forward primer, primer 16F27 (5h-AGAG- DNA base composition. The GjC content of DNA of strain TTTGATCMTGGCTCAG-3h), annealed at positions 8–27 S-30 was analysed in the same way as that from strains S-7, and the reverse primer, primer 16R1488 (5h-CGGTTACC- S-31T and S-36 (Bouchotroch et al., 1999). Exponential TTGTTAGGACTTCACC-3 ) (Pharmacia), annealed at the " h phase cells were ruptured with lysozyme (10 mg l− ) and SDS complement of positions 1511–1488 [E. coli numbering " (2n5gl− ) and their DNA was purified using the method of (Brosius et al., 1978)]. Marmur (1961). The DNA G C content was determined j Sequencing of PCR-amplified DNA. The PCR products were from the midpoint value (T ) of the thermal denaturation m purified using the Qiaquick spin gel extraction kit (Qiagen) profile (Marmur & Doty, 1962), obtained with a Perkin- according to the manufacturer’s recommendations. Direct Elmer UV-Vis Lambda3B spectrophotometer at 260 nm and " sequence determinations of PCR-amplified DNAs were programmed for temperature increases of 1 0 C min− . T n m m carried out with the ABI PRISM dye-terminator, cycle- was determined by the graphic method described by sequencing, ready-reaction kit (Perkin-Elmer) and an ABI Ferragut & Leclerc (1976) and the G C content was j PRISM 377 sequencer (Perkin-Elmer) according to the calculated from this temperature using the equation of Owen manufacturer’s instructions. & Hill (1979). The Tm value of reference DNA from Escherichia coli NCTC 9001 was taken to be 74n6 mCin0n1i Phylogenetic analysis. The sequences obtained (S-7, SSC (Owen & Pitcher, 1985). The means of at least two 1395 bp; S-30, 1398 bp; S-31T, 1396 bp; S-36, 1398 bp) independent determinations for each experiment were calcu- were compared to reference 16S rRNA gene sequences lated. available in the GenBank and EMBL databases obtained

1626 International Journal of Systematic and Evolutionary Microbiology 51 Halomonas maura sp. nov. from the National Center for Biotechnology Information washing in 0n1 M sodium cacodylate, 0n05% (w\v), ru- database using the  search. The 16S rDNA sequences thenium red was added for 4 h. The cells were dehydrated in were aligned with the ,   and  an ethanol series (30, 50, 70, 90 and 100%, v\v) at room programs and the alignments were corrected manually. temperature, each dehydration step taking 20 min. The Evolutionary distances, including a correction factor for samples were critical-point dried and sputter-coated with reverse mutations (Jukes & Cantor, 1969), were calculated gold. Electron micrographs were obtained by using a Zeiss for sequence pairs by using a ‘mask’ (Lane, 1991) for non- DSM950 scanning electron microscope (15 kV, beam speci- homologous or uncertain nucleotide positions. Dendro- men angle 45m). grams were generated by using a pair-wise, weighted, least- Salt requirements of strain S-31T. The growth rate of strain S- square distance method (Olsen, 1987). 31T was determined in MY medium with different salt Nucleotide sequence accession numbers. The EMBL ac- concentrations to establish optimum growth conditions. cession numbers for the 16S rDNA nucleotide sequences of The salt concentrations assayed ranged from 0n5to30n0% the reference strains used in the phylogenetic analyses are as (w\v) and were prepared from a mixture of sea salts follows: Aeromonas hydrophila ATCC 7966T, X60404; T (Rodrı!guez-Valera et al., 1981). To inoculate the media, Chromohalobacter marismortui ATCC 17056 , X87219; 0n1 ml from an 18 h culture (OD&#! of 1n0) grown in standard Chromohalobacter marismortui A-65, X87220; Halomonas T MY medium [7n5% (w\v) salts] was diluted appropriately aquamarina ATCC 14400 , M93352; Halomonas campisalis and added to 500 ml Erlenmeyer flasks containing 100 ml ATCC 700597T, AF054286; Halomonas cupida DSM 4740T, T MY each with the different salt concentrations. The cultures L42615; Halomonas desiderata DSM 9502 , X92417; were then incubated in a rotary shaker (100 r.p.m.) at 32 mC. Halomonas elongata ATCC 33173T, M93355; H. eurihalina T All experiments were carried out in triplicate. Plate counts ATCC 49336 , X87218; Halomonas halmophila ATCC were made using MY medium with the same salt con- 19717T, M59153; Halomonas halodurans DSM 5160T, T centration in each case. L42619; Halomonas halophila DSM 4770 , M93353; T Halomonas magadiensis NCIMB 13595T, X92150; Halo- Using similar methods, the question of whether strain S-31 monas marina ATCC 25374T, M93354; Halomonas required NaCl alone or whether it needed other salts for meridiana DSM 5425T, M93356; Halomonas pacifica DSM growth was analysed. The following salts or combination of 4742T, L42616; Halomonas pantelleriensis DSM 9661T, salts were tested: (a) 1n2 M NaCl; (b) 1n2 M NaClj0n13 M X93493; Halomonas salina ATCC 49509T, X87217; MgSO%.7H#O; (c) 1n2 M NaClj0n13 M MgCl#.6H#O; (d) Halomonas subglaciescola DSM 4683T, M93358; Halomonas 1n2 M NaClj0n07 M MgSO%.7H#Oj0n06 M MgCl#.6H#O; variabilis DSM 3051T, M93357; Halomonas venusta ATCC (e) 1n2 M NaClj0n07 M MgSO%.7H#Oj0n06 M MgCl#. 27125T, L42618; Marinobacter hydrocarbonoclasticus ATCC 6H#Oj0n02 M KCl; (f) 1n2 M NaClj0n07 M MgSO%. 49840T, X67022; Marinomonas vaga ATCC 27119T, X67025; 7H#Oj0n06 M MgCl#.6H#Oj0n02 M KClj0n035 M and Pseudomonas aeruginosa DSM 50071T, X06684. CaCl#.2H#O. Sea salts at 9% (w\v) (Rodrı!guez-Valera T et al., 1981) were used as control because it was the optimum Electron microscopy of strain S-31 salt concentration according to the above experiment. (a) Negative stain preparation. Carbon-coated Formvar grids Strain S-31T grew in those media containing NaCl and were placed face-down over a droplet of mid-exponential- MgSO .7H O (or MgCl .6H O), but not with NaCl as the phase culture in MY medium. After 4 min, the grid was % # # # sole salt. It did not need KCl or CaCl .2H O. Our next step, removed, blotted briefly with filter paper and, without # # therefore, was to decide upon the optimum concentrations drying, transferred to 0 05% (w\v) ruthenium red solution n of NaCl and MgSO .7H O needed for growth. The minimum (pH 7 2), then blotted quickly, air-dried and examined under % # n concentration of salts required both for growth and for the a Zeiss TEM EM 10C transmission electron microscope. prevention of cell lysis was also determined. (b) Ultrathin sections. Mid-exponential-phase cells were fixed in 2n5% (v\v) glutaraldehyde, 0n05% (w\v) ruthenium The specific requirement for NaCl was examined by sub- red, buffered with 0n05 M sodium cacodylate, 2 mM stituting other salts at concentrations of 1n2 M (optimum MgCl#.6H#O (pH 7n4) at 4 mC for 4 h. After washing in 0n1M NaCl concentration). In this way, KCl, LiCl, MgCl#.6H#O and NH%Cl were used to test for a specific Na+ requirement. sodium cacodylate, the cells were post-fixed with 1% (w\v) − OsO% in 0n1 M sodium cacodylate at room temperature for The Cl requirement was examined with NaBr, NaNO$, 1 h. The cells were dehydrated in an ethanol series (30, 50, Na#SO% and Na#S#O$. All media were supplemented with 70, 90 and 100%, v\v) at room temperature, each de- 0n2 M MgSO%.7H#O (optimum concentration). The results hydration step taking 20 min. Infiltration with an embedding were compared with those from a positive control contain- resin\ethanol mixture (1:1, v\v) was done overnight at ing optimum concentrations of both NaCl (1n2 M) and room temperature, followed by pure resin infiltration, also MgSO%.7H#O(0n2 M). overnight. Cells were transferred into a gelatin capsule and Fatty acid analysis of strain S-31T. This study was carried out filled with resin monomer. Polymerization was done at 60 mC by the Analytical Services of Microbial Identification for 8 h. Ultrathin sections were cut with a Reichert Ultracut Systems (Williston, VT 05495, USA), using the MID\ S ultramicrotome equipped with a diamond knife and then Hewlett Packard Microbial Identification System (MIS), collected on grids (300 mesh, Cu). The sections were stained which relies upon high-resolution GC to obtain the fatty for 10 min with 1% (w\v) aqueous uranyl acetate solution acid profile. and lead citrate. The samples were examined under a transmission electron microscope (Zeiss TEM EM 10C, 30 µm objective aperture, 80 kV acceleration voltage) and RESULTS photographed on Agfa Scientia EM film. DNA base composition and DNA–DNA hybridization (c) Scanning. Cells from mid-exponential-phase cultures were bound to poly--lysine-coated slides during the fixation The GjC content of strain S-30 was 62n2mol%, process, which was carried out overnight at 4 mCinan which was very similar to that of the other three atmosphere saturated with glutaraldehyde vapour. After strains.

International Journal of Systematic and Evolutionary Microbiology 51 1627 S. Bouchotroch and others

Table 1. DNA–DNA hybridizaton between strain S-31T Phylogenetic analysis and other related species of the genus Halomonas ...... The affiliation of the isolates with the genus Halomonas Values are mean results of at least three independent was confirmed by 16S rRNA gene sequence com- parisons. The almost complete 16S rRNA gene determinations, which generally did not differ by more than T 5%. T, Type strain. sequences of strains S-31 , S-7, S-30 and S-36 (about 1500 bases) were determined and were aligned with Organism Hybridization (%) those reported for the other species of Halomonas to reconstruct a phylogenetic tree. Sequences from re- S-31T 100n0 lated halophilic taxa and from non-halophilic bacteria S-30 77n4 were used as the outgroup. Positions with any deletion S-36 75n5 or of uncertain alignment were removed and the remaining positions were used. The 16S rDNA of S-7 80n8 T Halomonas halmophila ATCC 19717T 38n5 strains S-31 , S-7, S-30 and S-36 had more than 97% Halomonas halophila DSM 4770T 21n5 similarity. When compared to the reference strains, the Halomonas salina ATCC 49509T 29n3 highest levels of similarity were with the 16S rRNA Halomonas eurihalina ATCC 49336T 30n8 gene sequences of H. salina (94n5–95n9%),H. eurihalina Halomonas pacifica DSM 4742T 39n0 (95n2–96n6%), H. elongata (94n0–95n3%), H. halmo- Halomonas elongata ATCC 33173T 34n0 phila (94n1–95n6%) and H. pacifica (95n2–96n7%), all of which had ! 39% DNA–DNA homology (Table 1). Fig. 1 shows the general structure of the phylogenetic tree obtained from the similarity matrix. The phylo- The levels of relatedness between the labelled DNA genetic relationship between our isolates, the other from strain S-31T and DNA from the other isolates species belonging to the genus Halomonas and other T were 75n5–80n8% (Table 1). On the other hand, low taxa are represented. Strains S-31 , S-7, S-30 and S-36 levels of relatedness (21n5–39n0%) were found between are on the same phylogenetic branch and close to H. these four strains and the other type strains of elongata, H. eurihalina, H. pacifica and H. salina. Our phenotypically and phylogenetically related species phylogenetic analysis agrees with the DNA homology used for comparison. study and, overall, results clearly show that our isolates

T

...... Fig. 1. Phylogenetic relationships of four strains of Halomonas maura to other Halomonas species and to other taxa of Gram-negative halophilic and non-halophilic bacteria. Only bootstrap values above 60% are shown (1000 replications). Bar, 0n01 expected changes per site.

1628 International Journal of Systematic and Evolutionary Microbiology 51 Halomonas maura sp. nov.

in long filaments. They measured 7n2–8n6i0n6–0n7 µm. (a) Thin sections revealed a typical Gram-negative cell envelope profile (Fig. 2). The cells contained poly-β- hydroxyalkanoate (PHA) granules. EPS appeared associated with the cell surface, the polysaccharide material forming a thick layer around the bacterium. As with other moderately halophilic bacteria, inter- mediate salt concentrations were required for optimum growth. Strain S-31T did, however, prove to have euryhaline characteristics, being able to grow in a wide range of salt concentrations (between 1 and 15%, −" w\v). An optimum growth rate of 0n265 h was observed in the presence of 9% (w\v) sea salts, although the bacteria were also capable of growing with NaCl plus MgSO%.7H#O (or MgCl#.6H#O). An −" (b) optimum growth rate of 0n144 h was achieved with these two salts combined at concentrations of 1n2 and 0n2 M, respectively. The minimum concentrations of NaCl and MgCl#.6H#O required for growth were 0n2 and 0n04 M, respectively. Cell lysis occurred when they were suspended without NaCl, regardless of the # magnesium concentration. Mg + cations could not prevent lysis, which occurred even in the presence of 0n13 M MgSO%.7H#O. The bacteria had a strict and specific requirement for Na+, as no growth was observed in an osmotically equivalent medium which lacked Na+. Furthermore, − − the chloride ion could not be replaced by Br ,NO$, #− #− SO% or S#O$ .

(c) The predominant fatty acids detected were 18:1ω7c, 16:1ω7c\2-OH i15:0 and 16:0. These fatty acids accounted for more than 81% of the total detected in S-31T. The following fatty acids were also detected in minor proportions: 10:0; 3-OH 10:0; i11:0; 12:0; 3-OH 12:0; 14:0; a17:1ω9c; 17:0; 18:0; and cy19:0ω8c.

DISCUSSION The aim of this study was to classify four EPS- producing strains selected from a larger group of moderately halophilic, Gram-negative aerobic rods. These bacteria had previously been tentatively identified as Halomonas spp., although they were not ...... closely related to any of the hitherto recognized species Fig. 2. Electron micrographs of strain S-31T stained with belonging to this genus. On the other hand, they ruthenium red. (a) Transmission electron micrograph of the presented great phenotypic similarity amongst them- negatively stained strain. (b) Transmission electron micrograph selves, as was revealed by numerical analysis of their of an ultrathin section showing the PHA granules. (c) Scanning phenotypic attributes (Bouchotroch et al., 1999). These electron micrograph. Bars, 1 µm. strains produced EPS of considerable biotechnological interest (Bouchotroch et al., 2000). Phenotypically, our isolates differed in many respects are phylogenetically and genotypically different from from the rest of the species belonging to the genus previously described species of Halomonas. Halomonas. H. eurihalina is the only species of Halo- monas characterized by EPS production (Quesada et Cells from the moderately halophilic strain S-31T were al., 1993), but this species does not seem to be non-flagellated rods with rounded ends, character- phenotypically related to the novel isolates, as has istically appearing singly or in pairs, but occasionally been previously demonstrated (Bouchotroch et al.,

International Journal of Systematic and Evolutionary Microbiology 51 1629 S. Bouchotroch and others

Table 2. Main differences between Halomonas maura and other phenotypically related species of the genus Halomonas ...... Data from Ventosa et al. (1998) and this study. Strains: 1, Halomonas maura;2,Halomonas elongata;3,Halomonas eurihalina; 4, Halomonas halmophila;5,Halomonas halodenitrificans;6,Halomonas halodurans;7,Halomonas halophila;8,Halomonas salina; 9, Halomonas variabilis. , Not determined; j, positive; k, negative; , differs amongst organisms.

Character 1 2 3 4 5 6 7 8 9

Morphology Long rods Rods Short rods Rods Short rods Rods Rods Short rods Curved rods Size (µm) 0n5–0n7i6n0–9n0  0n8–1n0i2n0–2n50n3–0n6i0n9–1n30n5–0n9i0n9–1n20n4–0n6i1n5–2n00n5–0n7i1n5–2n00n7–0n8i2n0–2n50n5–0n8i1n0–3n0 Pigmentation Cream None Cream Cream Cream None Cream Yellowish or cream Cream EPS jk j k k k k k k Motility kj k j k j j k j Facultative kj k k k k k k k anaerobe Acid from glucose kj k j k j j k  Oxidase jj k j j j j j j NaCl range 1n0–15n03n5–20n05n0–20n00n5–20n03n0–20n03n5–20n02n0–30n02n5–20n07n0–28n0 (%, v\w) NaCl optimum 7n5–10n03n5–8n07n5  5n0–9n08n07n55n010n0 (%, v\w) pH range 6n0–9n05n0–9n05n0–10n0   5n5–8n55n0–10n06n0–10n06n5–8n4 Temperature 10–40 15–45 15–45  5–32 4–37 15–45 15–40 15–37 range ( mC) Nitrate reduction jj j k j k j j k Nitrite reduction kj k k j k k k k H#S production jk j  kkjj k Hydrolysis of: Casein kk k k   kk DNA kk j j   kk Aesculin kk j k k j j k j Gelatin k  jkkkkkk Tween 80 kk j k k  kk Habitat Saline soils Salterns Salterns, saline soils ead Sea Meat-curing brines Estuarine water Saline soils Salterns, saline soils Great Salt Lake GjC content 62n2–64n160n559n1–65n763n064n0–66n063n266n760n7–64n261n0 (mol%)

1999). The most notable differences between our Schleifer, 1999). Stackebrandt & Goebel (1994) state isolates and other phenotypically related species of that levels of similarity between 16S rRNA gene Halomonas are shown in Table 2. sequences lower than 97% suggest that the strains in question do not belong to the same species. On the Whole-cell-derived fatty acid patterns were essentially other hand, organisms with 70% or higher DNA similar and contained features indicative of the Halo- similarity usually have more than 97% sequence monadaceae family (Skerratt et al., 1991). Some minor identity. The 16S rRNA gene sequences of our strains differences were observed in their fatty acid com- T were highly similar and DNA similarity was above position: strain S-31 lacked cy17:0. 70%. The phylogenetic study revealed the highest level of similarity was with the 16S rRNA gene sequence of The DNA base composition of our isolates ranged H. salina (94 5–95 9%), H. eurihalina (95 2–96 6%), between 62 2 and 64 1 mol% (Bouchotroch et al., n n n n n n H. elongata (94 0–95 3%),H. halmophila (94 1–95 6%) 1999), which is within the range of values for members n n n n and H. pacifica (95 2–96 7%), whilst the rest of the of the genus Halomonas (Mellado et al., 1995). DNA n n Halomonas species showed considerably lower simi- relatedness experiments showed that the novel bacteria larity with the novel isolates. form a single DNA similarity group with DNA–DNA hybridization values higher than 75n5%. This result In summary, previous phenotypic data (Bouchotroch agrees with the high phenotypic similarity of the et al., 1999) and the results of this present study strains. Nevertheless, very low DNA–DNA hybrid- indicate that our strains should be classified as a novel ization was found between strain S-31T and the species, for which the name Halomonas maura sp. nov. other species of Halomonas. The highest DNA–DNA is proposed. relatedness value (39%) was found with H. pacifica, but this value is far below the consensus value of 70% Description of Halomonas maura sp. nov. for delineating a species (Stackebrandt & Goebel, Halomonas maura (mauhra. L. adj. maurus northwest 1994; Wayne et al., 1994). African). Since low DNA–DNA hybridization values do not Gram-negative rods, 6–9i0n5–0n7 µm, occurring provide significant information on phylogenetic either singly or in pairs, occasionally as long filaments. relationships, the next step of our study was to analyse Cells are capsulated, non-motile and accumulate PHA. our halophiles’ 16S rRNA gene sequence (Ludwig & Does not form endospores. Colonies on MY medium

1630 International Journal of Systematic and Evolutionary Microbiology 51 Halomonas maura sp. nov. are circular, convex, mucoid and cream-coloured; they at a ratio of 1:4:2n5. The EPS aqueous solution −" are 2–3 mm in diameter after 24 h, but reach more (1%, w\v) attains a viscosity of 10n98 Pa s at 4n5Pa than 5 mm in diameter and are considerably mucoid shear rate. EPS S-31 is able to emulsify crude oil more after 72 h at 32 mC. Moderate , capable of efficiently than some chemical surfactants such as growth in salt concentrations of between 1 and 15% Triton X-100 and Span 60. (w\v). Optimum growth occurs in 7n5–10n0% (w\v) sea salts. No growth occurs in the absence of salts. ACKNOWLEDGEMENTS Grows at temperatures between 10 and 40 mC (op- timum growth at 32 C) and at pH values between Financial support was provided by grants from the Spanish m Ministerio de Educacio! n y Cultura (BIO98-0897-C02-01) 6 and 9 (optimum growth at pH 7n2). Chemo- and from the Junta de Andalucı!a. The authors are very organotrophic. Metabolism is of the respiratory type, grateful to Dr R. Xalabarder and to SKW Biosystems SA with oxygen as the terminal acceptor. Capable of for testing EPS in their laboratories and partially financing anaerobic growth in the presence of nitrate, but not in their research. We thank Rafael Pa! ez, Ma Carmen Lie! bana, the presence of fumarate. Catalase and oxidase are Ma Jose! Sa! nchez, Juan Antonio Mata and Cristina Ruiz- produced. Does not produce acids from sugars. Sel- Garcı!a for their technical assistance and Concepcio! n enite is reduced and gluconate is oxidized. H#Sis Herna! ndez, David Porcel and Alicia Gonza! lez for their produced from peptones and Tween 20 is hydrolysed. expertise in the microscope studies. We also thank our Indole, methyl red and Voges–Proskauer are negative. colleague Dr J. Trout for revising our English text. Aesculin, tyrosine, DNA, gelatin, casein, urea, lecithin and Tween 80 are not hydrolysed. 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