HqiA, a novel quorum-quenching enzyme which expands the AHL lactonase family.

The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-acyl homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demonstrated that a single gene (hqiA) allowed AHL degradation. Enzymatic analysis using purified HqiA and HPLC/MS revealed that this protein has lactonase activity on a broad range of AHLs. The introduction of hqiA in the plant pathogen Pectobacterium carotovorum efficiently interfered with both the synthesis of AHLs and quorum-sensing regulated functions, such as swarming motility and the production of maceration enzymes. Bioinformatic analyses highlighted that HqiA showed no sequence homology with the known prototypic AHL lactonases or acylases, thus expanding the AHL-degrading enzymes with a new family related to the cysteine hydrolase (CHase) group. The complete sequence analysis of the fosmid showed that 31 ORFs out of the 46 identified were related to Deltaproteobacteria, whilst many intercalated ORFs presented high homology with other taxa. In this sense, hqiA appeared to be assigned to the Hyphomonas genus (Alphaproteobacteria), suggesting that horizontal gene transfer had occurred.

Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture.

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

Characterization of haloglycan, an exopolysaccharide produced by Halomonas stenophila HK30.

We have conducted a thorough study of the exopolysaccharide (EPS) produced by strain HK30 of Halomonas stenophila, which we have named haloglycan. This strain was chosen during an ongoing research programme aimed at finding novel exopolysaccharide-producing halophilic bacteria in unexplored hypersaline habitats. Strain HK30 was isolated from a saline-wetland in Brikcha (Morocco) and identified as belonging to the species H. stenophila. It produced EPS mainly during the exponential growth phase and to a lesser extent during the stationary phase. Culture parameters influenced both bacterial growth and EPS production, EPS yield always being directly related to the quantity of biomass. Under optimum culture conditions, strain HK30 produced 3.89 g of EPS per litre of medium. The polymer was a sulphated heteropolysaccharide composed of two fractions, with molecular masses of 8.2 × 10(4) and 1.4 × 10(6). The crude EPS contained 44 ± 0.1% w/w carbohydrates and the following monosaccharide composition: glucose (24 ± 1.73), glucuronic acid (7.5 ± 0.37), mannose (5.5 ± 0.17), fucose (4.5 ± 0.36), galactose (1.2 ± 0.17) and rhamnose (1 ± 0.05) (%, w/w). It produced solutions of high viscosity and pseudoplastic behaviour that showed interesting flocculating and emulsifying activities and was also involved in forming biofilm.

Diversity of halophilic bacteria isolated from Rambla Salada, Murcia (Spain).

In this study we analyzed the diversity of the halophilic bacteria community from Rambla Salada during the years 2006 and 2007. We collected a total of 364 strains, which were then identified by means of phenotypic tests and by the hypervariable V1-V3 region of the 16S rRNA sequences (around 500 bp). The ribosomal data showed that the isolates belonged to Proteobacteria (72.5%), Firmicutes (25.8%), Actinobacteria (1.4%), and Bacteroidetes (0.3%) phyla, with Gammaproteobacteria the predominant class. Halomonas was the most abundant genus (41.2% isolates) followed by Marinobacter (12.9% isolates) and Bacillus (12.6% isolates). In addition, 9 strains showed <97% sequence identity with validly described species and may well represent new taxa. The diversity of the bacterial community analyzed with the DOTUR package determined 139 operational taxonomic units at 3% genetic distance level. Rarefaction curves and diversity indexes demonstrated that our collection of isolates adequately represented all the bacterial community at Rambla Salada that can be grown under the conditions used in this work. We found that the sampling season influenced the composition of the bacterial community, and bacterial diversity was higher in 2007; this fact could be related to lower salinity at this sampling time.

Diversity and distribution of Halomonas in Rambla Salada, a hypersaline environment in the southeast of Spain.

We have studied the diversity and distribution of Halomonas populations in the hypersaline habitat Rambla Salada (Murcia, southeastern Spain) by using different molecular techniques. Denaturing gradient gel electrophoresis (DGGE) using specific primers for the 16S rRNA gene of Halomonas followed by a multivariate analysis of the results indicated that richness and evenness of the Halomonas populations were mainly influenced by the season. We found no significant differences between the types of samples studied, from either watery sediments or soil samples. The highest value of diversity was reached in June 2006, the season with the highest salinity. Furthermore, canonical correspondence analysis (CCA) demonstrated that both salinity and pH significantly affected the structure of the Halomonas community. Halomonas almeriensis and two denitrifiers, H. ilicicola and H. ventosae were the predominant species. CARD-FISH showed that the percentage of Halomonas cells with respect to the total number of microorganisms ranged from 4.4% to 5.7%. To study the functional role of denitrifying species, we designed new primer sets targeting denitrification nirS and nosZ genes. Using these primers, we analyzed sediments from the upwelling zone collected in June 2006, where we found the highest percentage of denitrifiers (74%). Halomonas ventosae was the predominant denitrifier in this site.

Draft Genome Sequence of the Moderately Halophilic Gammaproteobacterium Halomonas anticariensis FP35T.

Halomonas anticariensis strain FP35(T) is a moderately halophilic bacterium isolated from a soil sample taken from Fuente de Piedra, a saline wetland in the province of Málaga (Spain), which produces an exopolysaccharide and quorum-sensing signaling molecules of the type N-acylhomoserine lactone. We report here the draft genome sequence of this gammaproteobacterium.

Genetic and phenotypic analysis of the GacS/GacA system in the moderate halophile Halomonas anticariensis.

A multisensory, hybrid histidine kinase (HK) and a response regulator (RR), which together may well constitute a two-component regulatory system (TCS), have been located in Halomonas anticariensis FP35(T) by transposon mutagenesis. This TCS is homologous to the GacS/GacA system described for many Gram-negative bacteria. An analysis of crude N-acylhomoserine lactone (AHL) extracts from cultures of FP35gacS and FP35gacA mutants showed that they produced lower quantities of AHLs than the wild-type strain. In addition, RT-PCR analysis revealed a considerable decrease in the expression of the quorum-sensing (QS) genes hanR and hanI compared with the wild-type strain. This result indicates that the GacS/GacA TCS exerts a positive effect upon the QS HanR/HanI system and suggests its integral involvement in the intercellular communication strategies of this bacterium. We have also demonstrated the influence of GacS and GacA upon exopolysaccharide production and biofilm formation, in which this regulatory machinery appears to play a key role in an overall system that co-ordinates gene expression and behaviour in H. anticariensis FP35(T) in response to environmental conditions.

Molecular ecology techniques reveal both spatial and temporal variations in the diversity of archaeal communities within the athalassohaline environment of Rambla Salada, Spain.

We have studied the distribution of the archaeal communities in Rambla Salada (Murcia, Spain) over three different seasons and observed the influence upon them of the environmental variables, salinity, pH, oxygen and temperature. Samples were collected from three representative sites in order to gain an insight into the archaeal population of the rambla as a whole. Denaturing gradient gel electrophoresis patterns and diversity indexes indicate that the diversity of the archaeal community in Rambla Salada changed mainly according to the season. We found no significant differences between the types of sample studied: watery sediments and soils. The upwelling zone showed most diversity in its archaeal community. The overall archaeal community was composed mainly of Halobacteriales and Thermoplasmatales, accounting for 72.6 and 12.1 % of the total, respectively. Haloarcula was the most abundant genus, being present at all three sites during all three seasons. Some few Crenarchaeota were always found, mainly at low-salinity levels. Ordination canonical correspondence analysis demonstrated that salinity affected the structure of the community significantly, whilst pH, oxygen and temperature did so to a lesser extent. Most Halobacteriales correlated positively with salinity and pH, whilst Thermoplasmatales correlated negatively with both salinity and pH and positively with temperature and oxygen. The archaeal community with the highest diversity was sampled during June 2006, the season with the highest salt concentration. Catalyzed reporter deposition-fluorescence in situ hybridization showed that the percentage of archaea in Rambla Salada compared to the total number of microorganisms (as measured by DAPI) ranged from 11.1 to 16.7 %. Our research group had isolated the most abundant taxon, Haloarcula, previously in Rambla Salada using classical culture techniques, but on this occasion, using culture-independent methods, we were also able to identify some phylotypes, Halorubrum, Methanolobus, Natronomonas, Halomicrobium, Halobacterium, Halosimplex, uncultured Thermoplasmatales and uncultured Crenarchaeota, that had remained undetected during our earlier studies in this habitat.

Quorum sensing in some representative species of halomonadaceae.

Cell-to-cell communication, or quorum-sensing (QS), systems are employed by bacteria for promoting collective behaviour within a population. An analysis to detect QS signal molecules in 43 species of the Halomonadaceae family revealed that they produced N-acyl homoserine lactones (AHLs), which suggests that the QS system is widespread throughout this group of bacteria. Thin-layer chromatography (TLC) analysis of crude AHL extracts, using Agrobacterium tumefaciens NTL4 (pZLR4) as biosensor strain, resulted in different profiles, which were not related to the various habitats of the species in question. To confirm AHL production in the Halomonadaceae species, PCR and DNA sequencing approaches were used to study the distribution of the luxI-type synthase gene. Phylogenetic analysis using sequence data revealed that 29 of the species studied contained a LuxI homolog. Phylogenetic analysis showed that sequences from Halomonadaceae species grouped together and were distinct from other members of the Gammaproteobacteria and also from species belonging to the Alphaproteobacteria and Betaproteobacteria.

The potential biotechnological applications of the exopolysaccharide produced by the halophilic bacterium Halomonas almeriensis.

We have studied the extracellular polysaccharide (EPS) produced by the type strain, M8(T), of the halophilic bacterium Halomonas almeriensis, to ascertain whether it might have any biotechnological applications. All the cultural parameters tested influenced both bacterial growth and polysaccharide production. EPS production was mainly growth-associated and under optimum environmental and nutritional conditions M8(T) excreted about 1.7 g of EPS per litre of culture medium (about 0.4 g of EPS per gram of dry cell weight). Analysis by anion-exchange chromatography and high-performance size-exclusion chromatography indicated that the exopolysaccharide was composed of two fractions, one of 6.3 × 10(6) and another of 1.5 × 10(4) Daltons. The monosaccharide composition of the high-molecular-weight fraction was mannose (72% w/w), glucose (27.5% w/w) and rhamnose (0.5% w/w). The low-molecular-weight fraction contained mannose (70% w/w) and glucose (30% w/w). The EPS has a substantial protein fraction (1.1% w/w) and was capable of emulsifying several hydrophobic substrates, a capacity presumably related to its protein content. The EPS produced solutions of low viscosity with pseudoplastic behaviour. It also had a high capacity for binding some cations. It contained considerable quantities of sulphates (1.4% w/w), an unusual feature in bacterial polysaccharides. All these characteristics render it potentially useful as a biological agent, bio-detoxifier and emulsifier.

Halomonas ramblicola sp. nov., a moderately halophilic bacterium from Rambla Salada, a Mediterranean hypersaline rambla.

A moderately halophilic bacterium (strain RS-16(T)) was isolated from saline soil in Rambla Salada, a Mediterranean hypersaline rambla in Murcia, south-east Spain. Cells of strain RS-16(T) were Gram-negative rods, oxidase-negative and motile by peritrichous flagella. Strain RS-16(T) required NaCl for growth, and grew between 1% and 30% (w/v) NaCl (optimum, 5-7.5%), at temperatures of between 4 °C and 41 °C (optimum, 32-37 °C), and at pH values of between 5 and 10 (optimum, pH 7). Strain RS-16(T) was chemo-organotrophic and its metabolism was respiratory with oxygen and nitrate as terminal electron acceptors. It produced acids from d-glucose and myo-inositol, accumulated poly-ß-hydroxyalkanoate granules and produced cream colonies on MY 7.5% (w/v). The DNA G+C content of strain RS-16(T) was 56.2 mol%. A comparison of 16S rRNA gene sequences confirmed the relationship of strain RS-16(T) to species of the genus Halomonas. The most phylogenetically related species was Halomonas cerina SP4(T) (97.4%16S rRNA gene sequence similarity). In DNA-DNA hybridization assays strain RS-16(T) showed DNA-DNA relatedness values of 62.7 ± 3.09%, 64.5 ± 1.97% and 64.7 ± 1.74% to Halomonas cerina CECT 7282(T), Halomonas cerina CECT 7284 and Halomonas cerina CECT 7283, respectively. The major fatty acids of strain RS-16(T) were C(18:1)ω7c and C(16:0), and the predominant respiratory lipoquinone was ubiquinone, with nine isoprene units (Q-9). On the basis of these data, strain RS-16(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas ramblicola sp. nov. is proposed. The type strain is RS-16(T) ( = CECT 7896(T) = LMG 26647(T)).

The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium.

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35(T), which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI.

Halomonas stenophila sp. nov., a halophilic bacterium that produces sulphate exopolysaccharides with biological activity.

We have undertaken a polyphasic taxonomic study of two halophilic, Gram-negative bacterial strains, N12(T) and B-100, that produce sulphated exopolysaccharides with biological activity. They were isolated from two different saline soil samples. Both strains grow at NaCl concentrations within the range 3-15 % (w/v) [optimum 5-10 % (w/v)], at 15-37 °C (optimum 20-32 °C) and at pH 6-8 (optimum pH 7-8). Their 16S rRNA gene sequences indicate that they belong to the genus Halomonas in the class Gammaproteobacteria. Their closest relative is Halomonas nitroreducens, to which our strains show maximum 16S rRNA gene sequence similarity values of 98.7 % (N12(T)) and 98.3 % (B-100). Their DNA G+C contents are 61.9 and 63.8 mol%, respectively. The results of DNA-DNA hybridizations showed 43.9 % relatedness between strain N12(T) and H. nitroreducens CECT 7281(T), 30.5 % between N12(T) and Halomonas ventosae CECT 5797(T), 39.2 % between N12(T) and Halomonas fontilapidosi CECT 7341(T), 46.3 % between N12(T) and Halomonas maura CECT 5298(T), 52.9 % between N12(T) and Halomonas saccharevitans LMG 23976(T), 51.3 % between N12(T) and Halomonas koreensis JCM 12237(T) and 100 % between strains N12(T) and B-100. The major fatty acids of strain N12(T) are C(12 : 0) 3-OH (5.42 %), C(15 : 0) iso 2-OH/C(16 : 1)ω7c (17.37 %), C(16 : 0) (21.62 %) and C(18 : 1)ω7c (49.19 %). The proposed name for the novel species is Halomonas stenophila sp. nov. Strain N12(T) ( = CECT 7744(T)  = LMG 25812(T)) is the type strain.

Halomonas rifensis sp. nov., an exopolysaccharide-producing, halophilic bacterium isolated from a solar saltern.

A polyphasic taxonomic study was conducted on strain HK31(T), a moderately halophilic bacterium isolated from a solar saltern in Chefchaouen, Morocco. The strain was a Gram-reaction-negative, oxidase-positive rod, which was motile by means of peritrichous flagella. The strain required NaCl for growth and grew in salt concentrations (mixture of sea salts) of 0.5-20 % (w/v) (optimum 5-7.5 %, w/v), at 25-45 °C (optimum 32 °C) and at pH 5-10 (optimum pH 6-9). Strain HK31(T) did not produce acids from sugars and its metabolism was respiratory, using oxygen as terminal electron acceptor. The strain was positive for the accumulation of poly-ß-hydroxyalkanoate granules and formed mucoid colonies due to the excretion of an exopolysaccharide. The DNA G+C content was 61.5 mol%. 16S rRNA gene sequence analysis indicated that it belonged to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species was Halomonas anticariensis, with which strain HK31(T) showed a 16S rRNA gene sequence similarity of 96.48 %. Its major fatty acids were C(18 : 1)ω7c, C(16 : 0), C(19 : 0) cyclo ω8c, C(16 : 1)ω7c/iso-C(15 : 0) 2-OH and C(12 : 0) 3-OH and the predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q-9). Based on the evidence provided in this study, strain HK31(T) (= CECT 7698(T) = LMG 25695(T)) represents a novel species of the genus Halomonas, for which the name Halomonas rifensis is proposed.

An exopolysaccharide produced by the novel halophilic bacterium Halomonas stenophila strain B100 selectively induces apoptosis in human T leukaemia cells.

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria and have recently been attracting considerable attention from biotechnologists because of their potential applications in many fields, including biomedicine. We have screened the antitumoural activity of a panel of sulphated EPSs produced by a newly discovered species of halophilic bacteria. We found that the novel halophilic bacterium Halomonas stenophila strain B100 produced a heteropolysaccharide that, when oversulphated, exerted antitumoural activity on T cell lines deriving from acute lymphoblastic leukaemia (ALL). Only tumour cells were susceptible to apoptosis induced by the sulphated EPS (B100S), whilst primary T cells were resistant. Moreover, freshly isolated primary cells from the blood of patients with ALL were also susceptible to B100S-induced apoptosis. The newly discovered B100S is therefore the first bacterial EPS that has been demonstrated to exert a potent and selective pro-apoptotic effect on T leukaemia cells, and thus, we propose that the search for new antineoplastic drugs should include the screening of other bacterial EPSs, particularly those isolated from halophiles.

Characterization of the exopolysaccharide produced by Salipiger mucosus A3, a halophilic species belonging to the Alphaproteobacteria, isolated on the Spanish Mediterranean seaboard.

We have studied the exopolysaccharide produced by the type strain of Salipiger mucosus, a species of halophilic, EPS-producing (exopolysaccharide-producing) bacterium belonging to the Alphaproteobacteria. The strain, isolated on the Mediterranean seaboard, produced a polysaccharide, mainly during its exponential growth phase but also to a lesser extent during the stationary phase. Culture parameters influenced bacterial growth and EPS production. Yield was always directly related to the quantity of biomass in the culture. The polymer is a heteropolysaccharide with a molecular mass of 250 kDa and its components are glucose (19.7%, w/w), mannose (34%, w/w), galactose (32.9%, w/w) and fucose (13.4%, w/w). Fucose and fucose-rich oligosaccharides have applications in the fields of medicine and cosmetics. The chemical or enzymatic hydrolysis of fucose-rich polysaccharides offers a new efficient way to process fucose. The exopolysaccharide in question produces a solution of very low viscosity that shows pseudoplastic behavior and emulsifying activity on several hydrophobic substrates. It also has a high capacity for binding cations and incorporating considerable quantities of sulfates, this latter feature being very unusual in bacterial polysaccharides.

Denitrification as an important taxonomic marker within the genus Halomonas.

We have made a comprehensive study of the denitrifying species of the genus Halomonas, evaluating both the phylogenetic and phenotypic relationships amongst them and other species of Halomonas. The phylogenetic analysis was based on the 16S rRNA gene sequence as well as those of the three genes essential to a complete denitrification process: narH, nirS and nosZ. The main aim of the phenotypic study was to improve our knowledge of some of the species in question. To this end we investigated the type strain of each species, although in the case of Halomonas cerina we also studied strains R53 and 15CR. In addition to this we investigated some other strains phylogenetically related to Halomonas ventosae, Halomonas denitrificans and Halomonas koreensis that were isolated during this study. We also looked into the conditions under which all these bacteria denitrify. Our results indicate that these denitrifying species of Halomonas are all closely related. A numerical analysis of the phenotypic data demonstrates a high phenotypic similarity (73%) between most of them. In addition, all the denitrifying strains have a high G+C content of between 63 and 74.3 mol%. The results of the phylogenetic study point to two evolutionary lineages for the process. Although phenotypic similarity does not always reflect phylogenetic relatedness, we have found significant congruence between both features in Halomonas, making it clear that denitrifying ability should be considered as an important phenotypic and phylogenetic discriminatory marker within this genus.

Halomonas fontilapidosi sp. nov., a moderately halophilic, denitrifying bacterium.

We have made a polyphasic taxonomic study of strain 5CR(T), isolated from Fuente de Piedra, Málaga, southern Spain. The strain is a moderately halophilic, Gram-negative rod, oxidase-positive and motile by a single polar flagellum. It does not produce acids from sugars and shows respiratory metabolism, using oxygen, nitrate and nitrite as terminal electron acceptors. It requires NaCl and grows best with 5-7.5 % w/v at temperatures of between 32 and 45 degrees C within a pH range of 6-8. Its 16S rRNA gene sequence indicates that strain 5CR(T) belongs to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas alimentaria, H. nitroreducens, H. shengliensis and H. ventosae, with the type strains of which our strain showed 16S rRNA gene sequence similarity values of 96.7-97.8 %. DNA-DNA hybridization studies between strain 5CR(T) and H. ventosae CECT 5797(T), the phylogenetically nearest type strain, showed 40 % relatedness. Its G+C content is 65.7 mol%. Its major fatty acids are C(18 : 1)omega7c (31.36 %), C(16 : 0) (25.55 %), C(16 : 1)omega7c/iso-C(15 : 0) 2-OH (23.23 %), C(19 : 0) cyclo omega8c (8.14 %), C(12 : 0) 3-OH (5.76 %) and C(10 : 0) (2.22 %) and the predominant respiratory lipoquinone is ubiquinone with nine isoprene units (Q-9). The proposed name for the novel species is Halomonas fontilapidosi sp. nov., strain 5CR(T) (=CECT 7341(T) =LMG 24455(T)) being the type strain.

Halomonas cerina sp. nov., a moderately halophilic, denitrifying, exopolysaccharide-producing bacterium.

Three bacterial strains were isolated from different saline soils in Spain. The novel strains were moderately halophilic, exopolysaccharide-producing, Gram-negative, non-motile rods. The strains required NaCl and grew best with 7.5-10 % (w/v) NaCl in the medium. They formed wax-coloured colonies, were oxidase-positive and showed respiratory metabolism, using oxygen, nitrate and nitrite as terminal electron acceptors. The novel strains were able to denitrify and did not produce acid from sugars. The DNA G+C contents varied between 62.7 and 66.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and sequence signatures of this gene showed that all three novel isolates belonged to the genus Halomonas in the class Gammaproteobacteria and formed an independent phylogenetic line. The most phylogenetically related species were Halomonas alimentaria, Halomonas campaniensis, Halomonas gudaonensis and Halomonas ventosae, with which the novel strains showed 16S rRNA gene sequence similarity values of between 96.3 and 95.2 %. The principal fatty acids of the novel strains were 16 : 0, 18 : 1 omega 7c, 16 : 1 omega 7c and 19 : 0 cyclo omega 8c. The predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q-9). The name Halomonas cerina sp. nov. is proposed for these isolates. The type strain is SP4T (=CECT 7282T=LMG 24145T).

Halomonas nitroreducens sp. nov., a novel nitrate- and nitrite-reducing species.

We have carried out a polyphasic taxonomic study of strain 11ST, a halophilic, Gram-negative bacterium that is able to respire on nitrate and nitrite in anaerobiosis. Strain 11ST was isolated from a solar saltern in Cahuil, a region next to Pichilemu (Chile). It grows at NaCl concentrations within the range of 3-20 % w/v (optimum 5-7.5 %), temperatures from 4 to 45 degrees C (optimum 20-32 degrees C) and within a pH range of 5-10 (optimum pH 7-9). Its 16S rRNA gene sequence indicates that it belongs to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas alimentaria, H. denitrificans, H. organivorans and H. ventosae, with the type strains of which our strain showed maximum 16S rRNA gene sequence similarity values of 97.1-98.1 %. Its G+C content is 65.3 mol%. DNA-DNA hybridization studies showed 54.2 % relatedness between strain 11ST and H. alimentaria DSM 15356T and 47.2 % relatedness between strain 11ST and H. organivorans CECT 5995T. Lower DNA-DNA hybridization percentages were obtained against the type strains of other related Halomonas species. Its major fatty acids are C12 : 0 3-OH (5.56 %), iso-C15 : 0 2-OH/C16 : 1 omega 7c (22.30 %), C16 : 0 (27.80 %) and C18 : 1 omega 7c (29.92 %). The proposed name for the novel species is Halomonas nitroreducens sp. nov., with strain 11ST (=CECT 7281T =LMG 24185T) being the type strain.