Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia.

A Gram-negative, motile, rod-shaped bacteria, designated D7T, was isolated by using the dilution-to-extinction method, from a soil sample taken from Rambla Salada (Murcia, Spain). Growth of strain D7T was observed at 15-40 °C (optimum, 37 °C), pH 5-9 (optimum, 7) and 0-7.5% (w/v) NaCl (optimum, 3%). It is facultatively anaerobic. Phylogenetic analysis based on 16S rRNA gene sequence showed it belongs to the genus Marinobacterium. The in silico DDH and ANI against closest Marinobacterium relatives support its placement as a new species within this genus. The major fatty acids of strain D7T were C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The polar lipid profile consists of phosphatidylethanolamine, phosphatidylglycerol and two uncharacterized lipids. Ubiquinone 8 was the unique isoprenoid quinone detected. The DNA G + C content was 59.2 mol%. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characterization, strain D7T (= CECT 9818T = LMG 31312T) represents a novel species of the genus Marinobacterium for which the name Marinobacterium ramblicola sp. nov. is proposed. Genome-based metabolic reconstructions of strain D7T suggested a heterotrophic and chemolitotrophic lifestyle, as well as the capacity to biosynthetize and catabolize compatible solutes, and to degrade hydrocarbon aromatic compounds.

Biological Control of Verticillium Wilt on Olive Trees by the Salt-Tolerant Strain Bacillus velezensis XT1.

Verticillium wilt, caused by the pathogen Verticillium dahliae, is extremely devastating to olive trees (Olea europea). Currently, no successful control measure is available against it. The objective of this work was to evaluate the antifungal activity of Bacillus velezensis XT1, a well-characterized salt-tolerant biocontrol strain, against the highly virulent defoliating V. dahliae V024. In vitro, strain XT1 showed to reduce fungal mycelium from 34 to 100%, depending on if the assay was conducted with the supernatant, volatile compounds, lipopeptides or whole bacterial culture. In preventive treatments, when applied directly on young olive trees, it reduced Verticillium incidence rate and percentage of severity by 54 and ~80%, respectively. It increased polyphenol oxidase (PPO) activity by 395%, indicating an enhancement of disease resistance in plant tissues, and it decreased by 20.2% the number of fungal microsclerotia in soil. In adult infected trees, palliative inoculation of strain XT1 in the soil resulted in a reduction in Verticillium symptom severity by ~63%. Strain XT1 is biosafe, stable in soil and able to colonize olive roots endophytically. All the traits described above make B. velezensis XT1 a promising alternative to be used in agriculture for the management of Verticillium wilt.

Crop Protection against Botrytis cinerea by Rhizhosphere Biological Control Agent Bacillus velezensis XT1.

This study aims to evaluate the use of Bacillus velezensis strain XT1 as a plant growth-promoting rhizobacterium (PGPR) and biocontrol agent against B. cinerea in tomato and strawberry plants. Foliar and radicular applications of strain XT1 increased plant total biomass as compared to the control and B. cinerea-infected plants, with root applications being, on the whole, the most effective mode of treatment. Applications of the bacterium were found to reduce infection parameters such as disease incidence and severity by 50% and 60%, respectively. We analyzed stress parameters and phytohormone content in order to evaluate the capacity of XT1 to activate the defense system through phytohormonal regulation. Overall, the application of XT1 reduced oxidative damage, while the H2O2 and malondialdehyde (MDA) content was lower in XT1-treated and B. cinerea-infected plants as compared to non-XT1-treated plants. Moreover, treatment with XT1 induced callose deposition, thus boosting the response to pathogenic infection. The results of this study suggest that the signaling and activation pathways involved in defense mechanisms are mediated by jasmonic acid (JA) and ethylene hormones, which are induced by preventive treatment with XT1. The study also highlights the potential of preventive applications of strain XT1 to activate defense mechanisms in strawberry and tomato plants through hormone regulation.

Plant growth-promoting activity and quorum quenching-mediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6.

Given the major threat of phytopathogenic bacteria to food production and ecosystem stability worldwide, novel alternatives to conventional chemicals-based agricultural practices are needed to combat these bacteria. The objective of this study is to evaluate the ability of Pseudomonas segetis strain P6, which was isolated from the Salicornia europaea rhizosphere, to act as a potential biocontrol agent given its plant growth-promoting (PGP) and quorum quenching (QQ) activities. Seed biopriming and in vivo assays of tomato plants inoculated with strain P6 resulted in an increase in seedling height and weight. We detected QQ activity, involving enzymatic degradation of signal molecules in quorum sensing communication systems, against a broad range of N-acylhomoserine lactones (AHLs). HPLC-MRM data and phylogenetic analysis indicated that the QQ enzyme was an acylase. The QQ activity of strain P6 reduced soft rot symptoms caused by Dickeya solani, Pectobacterium atrosepticum and P. carotovorum on potato and carrot. In vivo assays showed that the PGP and QQ activities of strain P6 protect tomato plants against Pseudomonas syringae pv. tomato, indicating that strain P6 could have biotechnological applications. To our knowledge, this is the first report to show PGP and QQ activities in an indigenous Pseudomonas strain from Salicornia plants.

Psychrobacillus vulpis sp. nov., a new species isolated from faeces of a red fox in Spain.

A facultative anaerobic, chemoheterotrophic, endospore-forming, Gram-stain-positive rod, designated as strain Z8T, was isolated from red fox (Vulpes vulpes) faeces sampled at Tablas de Daimiel National Park, Ciudad Real, Spain. Strain Z8T grew at 0-37 °C (optimum, 28 °C), in the presence of 0-5.5 % (w/v) NaCl (2.5 %, w/v) and at pH 6-10 (pH 7). The strain was motile and positive for catalase, oxidase, H2S and siderophore production, acid and alkaline phosphatases, and N-acetylglucosamine, adipic acid and malate assimilation. It hydrolysed starch, DNA, l-tyrosine, Tween 20, Tween 80 and lecithovitellin. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Z8T is a member of the genus Psychrobacillus, showing high sequence similarity to Psychrobacillus lasiicapitis NEAU-3TGS17T (99.2 %) and Psychrobacillus soli NHI-2TT (99.1 %), and around 98 % to other known species of the genus Psychrobacillus. Digital DNA-DNA hybridization and average nucleotide identity values were lower than 24 and 79 %, respectively, with the most related species. In silico G+C content was 35.9 mol%. The major cellular fatty acids of strain Z8T were iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. The novel strain contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol as predominant polar lipids, and the main respiratory isoprenoid quinone was MK-8. Based on the 16S rRNA phylogenetic analysis, together with MLSA (recA, rpoB and gyrB), phylogenomic, chemotaxonomic and phenotypic results, we demonstrate that strain Z8T represents a novel species of the genus Psychrobacillus, for which the name Psychrobacillus vulpis sp. nov., is proposed. The type strain is Z8T (=CECT 9721T=LMG 31001T).

Silencing of Phytopathogen Communication by the Halotolerant PGPR Staphylococcus equorum Strain EN21.

Increasing world food demand together with soil erosion and indiscriminate use of chemical fertilization highlight the need to adopt sustainable crop production strategies. In this context, a combination of plant growth-promoting rhizobacteria (PGPR) and pathogen management represents a sustainable and efficient alternative. Though little studied, halophilic and halotolerant PGPR could be a beneficial plant growth promotion strategy for saline and non-saline soils. The virulence of many bacterial phytopathogens is regulated by quorum sensing (QS) systems. Quorum quenching (QQ) involves the enzymatic degradation of phytopathogen-generated signal molecules, mainly N-acyl homoserine lactones (AHLs). In this study, we investigate plant growth-promoting (PGP) activity and the capacity of the halotolerant bacterium Staphylococcus equorum strain EN21 to attenuate phytopathogens virulence through QQ. We used biopriming and in vivo tomato plant experiments to analyse the PGP activity of strain EN21. AHL inactivation was observed to reduce Pseudomonas syringae pv. tomato infections in tomato and Arabidopsis plants. Our study of Dickeya solani, Pectobacterium carotovorum subsp. carotovorum and Erwinia amylovora bacteria in potato tubers, carrots and pears, respectively, also demonstrated the effectiveness of QS interruption by EN21. Overall, this study highlights the potential of strain S. equorum EN21 in plant growth promotion and QQ-driven bacterial phytopathogen biocontrol.

Paenibacillus lutrae sp. nov., A Chitinolytic Species Isolated from A River Otter in Castril Natural Park, Granada, Spain.

A highly chitinolytic facultative anaerobic, chemoheterotrophic, endospore-forming, Gram-stain-positive, rod-shaped bacterial strain N10T was isolated from the feces of a river otter in the Castril Natural Park (Granada, Spain). It is a slightly halophilic, motile, catalase-, oxidase-, ACC deaminase- and C4 and C8 lipase-positive strain. It is aerobic, respiratory and has a fermentative metabolism using oxygen as an electron acceptor, produces acids from glucose and can fix nitrogen. Phylogenetic analysis of the 16S rRNA gene sequence, multilocus sequence analysis (MLSA) of 16S rRNA, gyrB, recA and rpoB, as well as phylogenomic analyses indicate that strain N10T is a novel species of the genus Paenibacillus, with the highest 16S rRNA sequence similarity (95.4%) to P. chitinolyticus LMG 18047T and <95% similarity to other species of the genus Paenibacillus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANIb) were 21.1% and <75%, respectively. Its major cellular fatty acids were anteiso-C15:0, C16:0, and iso-C15:0. G + C content ranged between 45%-50%. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with chemotaxonomic and phenotypic data, we demonstrate that type strain N10T (= CECT 9541T =LMG 30535T) is a novel species of genus Paenibacillus and the name Paenibacillus lutrae sp. nov. is proposed.

Study of Bacterial Community Composition and Correlation of Environmental Variables in Rambla Salada, a Hypersaline Environment in South-Eastern Spain.

We studied the bacterial community in Rambla Salada in three different sampling sites and in three different seasons and the effect of salinity, oxygen, and pH. All sites samples had high diversity and richness (Rr > 30). The diversity indexes and the analysis of dendrograms obtained by DGGE fingerprint after applying Pearson's and Dice's coefficient showed a strong influence of sampling season. The Pareto-Lorenz (PL) curves and Fo analysis indicated that the microbial communities were balanced and despite the changing environmental conditions, they can preserve their functionality. The main phyla detected by DGGE were Bacteroidetes (39.73%), Proteobacteria (28.43%), Firmicutes (8.23%), and Cyanobacteria (5.14%). The majority of the sequences corresponding to uncultured bacteria belonged to Bacteroidetes phylum. Within Proteobacteria, the main genera detected were Halothiobacillus and Roseovarius. The environmental factors which influenced the community in a higher degree were the salinity and oxygen. The bacteria belonging to Bacteroidetes and Proteobacteria were positively influenced by salinity. Nevertheless, bacteria related to Alpha- and Betaproteobacteria classes and phylum Firmicutes showed a positive correlation with oxygen and pH but negative with salinity. The phylum Cyanobacteria were less influenced by the environmental variables. The bacterial community composition of Rambla Salada was also studied by dilution-to-extinction technique. Using this method, 354 microorganisms were isolated. The 16S sequences of 61 isolates showed that the diversity was very different to those obtained by DGGE and with those obtained previously by using classic culture techniques. The taxa identified by dilution-to-extinction were Proteobacteria (81.92%), Firmicutes (11.30%), Actinobacteria (4.52%), and Bacteroidetes (2.26%) phyla with Gammaproteobacteria as predominant class (65.7%). The main genera were: Marinobacter (38.85%), Halomonas (20.2%), and Bacillus (11.2%). Nine of the 61 identified bacteria showed less than 97% sequence identity with validly described species and may well represent new taxa. The number of bacteria in different samples, locations, and seasons were calculated by CARD-FISH, ranging from 54.3 to 78.9% of the total prokaryotic population. In conclusion, the dilution-to-extinction technique could be a complementary method to classical culture based method, but neither gets to cultivate the major taxa detected by DGGE. The bacterial community was influenced significantly by the physico-chemical parameters (specially the salinity and oxygen), the location and the season of sampling.

Antifungal Activity of Lipopeptides From Bacillus XT1 CECT 8661 Against Botrytis cinerea.

This work aims to explore the capacity of a Bacillus methylotrophicus (later heterotypic synonym of Bacillus velezensis) strain named XT1 CECT 8661 against the necrotrophic plant pathogen Botrytis cinerea and to identify the compounds responsible for its activity. Q_TOF electrospray mass spectrometry analysis allows us to detect several lipopeptides - surfactin, bacillomycin, and fengycin - in XT1 cultures. In vitro antibiosis studies demonstrated the efficiency of the lipopeptide fraction for the inhibition of fungal growth. In fact, microscopy studies (SEM/TEM) revealed, an alteration of the morphology of the phytopathogen in interaction with lipopeptides, with resistance structures appearing in the early stages of growth of the fungus. Our studies, carried out with tomatoes, grapes, and strawberries have demonstrated the efficiency of Bacillus XT1 CECT 8661 lipopeptides against B. cinerea infection and it capability to trigger the antioxidant activity in fruit. Overall, the results of this study highlight the potential of lipopeptides of this strain as an effective biological control agent against the colonisation of B. cinerea.

Aphicidal Activity of Surfactants Produced by Bacillus atrophaeus L193.

The biosurfactants produced by Bacillus atrophaeus L193 was examined by their use in the control of the aphid Rhopalosiphum padi in order to suggest a friendly alternative to chemical pesticides. A screening of different culture media demonstrated the highest biosurfactant production by L193 in TSB supplemented with colloidal chitin. Surfactants, which are produced in large quantities (2.04 g/L), reduce surface tension to 33 mN/m. Electrospray Q-TOFS MS analysis demonstrated that lipopeptides, such as surfactins, fengycins, bacillomycins and iturins, are the predominant metabolites present in biosurfactants produced by strain L193. Treatment with L193 surfactants led to an aphid mortality rate of 59.8% within 24 h. Microscopy analysis showed that these compounds caused insect death by affecting cuticle membranes. An evaluation of aphid feeding activity also demonstrated that aphid feeding capacity is affected by treatment with surfactants. Moreover, microbial cultures of strain L193 and their supernatants also showed high levels of activity against R. padi, which is probably due to the presence of surfactants and hydrolytic enzymes such as proteases and glucanases. This study demonstrates that B. atrophaeus L193 is an effective treatment for plants affected by aphids.

Blastomonas quesadae sp. nov., isolated from a saline soil by dilution-to-extinction cultivation.

We isolated a Gram-stain-negative, aerobic bacterial strain, 912T, from a soil sample taken from Rambla Salada (Murcia), south-eastern Spain, by using the dilution-to-extinction method. Cells of the strain were motile with a polar flagellum, short rod-shaped, catalase- and oxidase-positive and grew at NaCl concentrations within the range 0-5 % (w/v) (optimum 3 %, w/v), at 4-32 °C (optimum 30 °C) and at pH 6-9 (optimum pH 7); bacteriochlorophyll a was produced. Analysis of the 16S rRNA gene sequence indicated that this strain belonged to the genus Blastomonas in the class Alphaproteobacteria. Its closest relatives were Blastomonas natatoria EY 4220T, Blastomonas ursincola KR-99T and Blastomonas aquatica PE 4-5T, to which the strain showed 16S rRNA gene sequence similarity values of 95.9, 95.8 and 95.1 %, respectively. The DNA G+C content was 63 mol%. The major fatty acids of strain 912T were C18 : 1ω7c/C18 : 1ω6c, C16 : 1ω7c/C16 : 1ω6c, C16 : 0 and C17 : 1ω6c. The predominant isoprenoid quinone was ubiquinone 10 (Q-10). The polar lipids contained diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphoglycolipid, one unidentified phospholipid and two unidentified polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Blastomonas, for which the name Blastomonas quesadae sp. nov. is proposed. Strain 912T (=CECT 9186T=LMG 29921T) is the type strain.

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.

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.

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)).

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.

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.

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.