Potential of the quorum-quenching and plant-growth promoting halotolerant Bacillus toyonensis AA1EC1 as biocontrol agent.

The use of fertilizers and pesticides to control plant diseases is widespread in intensive farming causing adverse effects together with the development of antimicrobial resistance pathogens. As the virulence of many Gram-negative phytopathogens is controlled by N-acyl-homoserine lactones (AHLs), the enzymatic disruption of this type of quorum-sensing (QS) signal molecules, mechanism known as quorum quenching (QQ), has been proposed as a promising alternative antivirulence therapy. In this study, a novel strain of Bacillus toyonensis isolated from the halophyte plant Arthrocaulon sp. exhibited numerous traits associated with plant growth promotion (PGP) and degraded a broad range of AHLs. Three lactonases and an acylase enzymes were identified in the bacterial genome and verified in vitro. The AHL-degrading activity of strain AA1EC1 significantly attenuated the virulence of relevant phytopathogens causing reduction of soft rot symptoms on potato and carrots. In vivo assays showed that strain AA1EC1 significantly increased plant length, stem width, root and aerial dry weights and total weight of tomato and protected plants against Pseudomonas syringae pv. tomato. To our knowledge, this is the first report to demonstrate PGP and QQ activities in the species B. toyonensis that make this strain as a promising phytostimulant and biocontrol agent.

Microbial composition of Saharan dust plumes deposited as red rain in Granada (Southern Spain).

During durst storms, also biological material is transported from arid areas such as the Sahara Desert. In the present work, rain samples containing significant amounts of mineral dust have been collected in Granada during different red rain episodes. Biological features (bacteria, biofilm, pollen grain and fungal spore) as well as size-particle distribution and mineralogical composition were studied by SEM. Nanobacteria were observed for the first time in red rain samples. A preliminary metabarcoding analysis was performed on three red rain samples. Here, Bacillota made up 18 % and Pseudomonadota 23 % of the whole prokaryotic community. The fungal community was characterized by a high abundance of Ascomycota and, dependent on the origin, the presence of Chytridiomycota. By means of 16S rRNA sequencing, 18 cultivable microorganisms were identified. In general, members of the phyla Pseudomonadota and Bacillota made up the majority of taxa. Some species, such as Peribacillus frigoritolerans and Bacillus halotolerans were isolated during three different red rain episodes. Generally, red rain carries a wide variety of microorganisms, being their ecosystem and health effects largely unknown.

Corrigendum: Identification of volatile organic compounds in extremophilic bacteria and their effective use in biocontrol of postharvest fungal phytopathogens.

[This corrects the article DOI: 10.3389/fmicb.2021.773092.].

Enhanced NSAIDs Solubility in Drug-Drug Formulations with Ciprofloxacin.

Drug-drug salts are a kind of pharmaceutical multicomponent solid in which the two co-existing components are active pharmaceutical ingredients (APIs) in their ionized forms. This novel approach has attracted great interest in the pharmaceutical industry since it not only allows concomitant formulations but also has proved potential to improve the pharmacokinetics of the involved APIs. This is especially interesting for those APIs that have relevant dose-dependent secondary effects, such as non-steroidal anti-inflammatory drugs (NSAIDs). In this work, six multidrug salts involving six different NSAIDs and the antibiotic ciprofloxacin are reported. The novel solids were synthesized using mechanochemical methods and comprehensively characterized in the solid state. Moreover, solubility and stability studies, as well as bacterial inhibition assays, were performed. Our results suggest that our drug-drug formulations enhanced the solubility of NSAIDs without affecting the antibiotic efficacy.

Peribacillus castrilensis sp. nov.: A Plant-Growth-Promoting and Biocontrol Species Isolated From a River Otter in Castril, Granada, Southern Spain.

A strictly aerobic, chemoheterotrophic, endospore-forming, Gram-positive, rod-shaped bacterial strain N3T was isolated from the feces of a river otter in Castril (Granada, southern Spain). It is halotolerant, motile, and catalase-, oxidase-, ACC deaminase-, and C4- and C8-lipase-positive. It promotes tomato plant growth and can reduce virulence in Erwinia amylovora CECT 222T and Dickeya solani LMG 25993T through interference in their quorum-sensing systems, although other antagonistic mechanisms could also occur. A phylogenetic analysis of the 16S rRNA gene sequence as well as the phenotypic and phylogenomic analyses indicated that the strain N3T is a novel species of the genus Peribacillus, with the highest 16S rRNA sequence similar to that of Bacillus frigoritolerans DSM 8801T (99.93%) and Peribacillus simplex DSM 1321T (99.80%). Genomic digital DNA-DNA hybridization (dDDH) between the strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 12.8 and 69.1%, respectively, and the average nucleotide identity (ANIb) of strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 67.84 and 93.21%, respectively. The genomic G + C content was 40.3 mol%. Its main cellular fatty acids were anteiso-C15:0 and iso-C15:0. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with the chemotaxonomic and phenotypic data, we demonstrated that the type strain N3T (=CECT 30509T = LMG 32505T) is a novel species of the genus Peribacillus and the name Peribacillus castrilensis sp. nov. is proposed.

Identification of Volatile Organic Compounds in Extremophilic Bacteria and Their Effective Use in Biocontrol of Postharvest Fungal Phytopathogens.

Phytopathogenic fungal growth in postharvest fruits and vegetables is responsible for 20-25% of production losses. Volatile organic compounds (VOCs) have been gaining importance in the food industry as a safe and ecofriendly alternative to pesticides for combating these phytopathogenic fungi. In this study, we analysed the ability of some VOCs produced by strains of the genera Bacillus, Peribacillus, Pseudomonas, Psychrobacillus and Staphylococcus to inhibit the growth of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Monilinia fructicola, Monilinia laxa and Sclerotinia sclerotiorum, in vitro and in vivo. We analysed bacterial VOCs by using GC/MS and 87 volatile compounds were identified, in particular acetoin, acetic acid, 2,3-butanediol, isopentanol, dimethyl disulphide and isopentyl isobutanoate. In vitro growth inhibition assays and in vivo experiments using cherry fruits showed that the best producers of VOCs, Bacillus atrophaeus L193, Bacillus velezensis XT1 and Psychrobacillus vulpis Z8, exhibited the highest antifungal activity against B. cinerea, M. fructicola and M. laxa, which highlights the potential of these strains to control postharvest diseases. Transmission electron microscopy micrographs of bacterial VOC-treated fungi clearly showed antifungal activity which led to an intense degeneration of cellular components of mycelium and cell death.

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.

Roseovarius bejariae sp. nov., a moderately halophilic bacterium isolated from a hypersaline steep-sided river bed.

An aerobic, Gram-stain-negative ovoid, designated as strain A21T, was isolated using the dilution-to-extinction method from a soil sample taken from Rambla Salada, an athalassohaline habitat located in Murcia (south-eastern Spain). Strain A21T is non-motile, has a respiratory metabolism and grows at NaCl concentrations within the range 0.5-15 % (w/v) [optimum, 5 % (w/v)], at 5-35 °C (optimum, 28 °C) and at pH 6-8 (optimum, pH 7.0). This strain is positive for catalase activity, oxidase activity and nitrate reduction. The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. The most closely related species are Roseovarius pacificus and Roseovarius halotolerans to which the strain A21T shows 16S rRNA gene sequence similarity values of 98.06 and 97.7 %, respectively. The average nucleotide identity in blast and digital DNA-DNA hybridization values between strain A21T and R. pacificus LMG 24575T are 76.8 and 21 %, respectively. The DNA G+C content based on the genome is 61.28 mol%. The major fatty acids (>5 % of the total fatty acids) of strain A21T are C18 : 1 ω7c/C18 : 1 ω6c and C16 : 0. The only detected isoprenoid quinone in strain A21T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and three unidentified polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius bejariae sp. nov. is proposed. Strain A21T (=CECT 9817T=LMG 31311T) is the type strain.

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.

Roseovarius ramblicola sp. nov., a moderately halophilic bacterium isolated from saline soil in Spain.

Strain D15T was isolated from a soil sample taken from Rambla Salada (Murcia), south-eastern Spain, by using the dilution-to-extinction method. The strain, a Gram-stain-negative aerobic bacteria, is non-motile, ovoid- or rod-shaped, catalase- and oxidase-positive, and grows at NaCl concentrations within the range 0.5-10 % (w/v) [optimum 3 % (w/v)], at 5-30 °C (optimum 28 °C) and at pH 6-9 (optimum pH 7.0). The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. Its closest relatives are Roseovarius tolerans EL-172T and Roseovarius azorensis SSW084T, to which the strain shows 16S rRNA gene-sequence similarity values of 96.1 and 95.3 %, respectively. The DNA G+C content is 63 mol%. The major fatty acids (>5 % of the total fatty acids) of strain D15T are C18 : 1ω7c, C16 : 0 and C12 : 0. The only detected isoprenoid quinone of strain D15T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, aminolipid and three polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius ramblicola sp. nov. is proposed. Strain D15T (=CECT 9424=LMG 30322) is the type strain.

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.

Idiomarina aquatica sp. nov., a moderately halophilic bacterium isolated from salterns.

Four bacterial strains, SN-14T, SN-4, M6-46 and M6-58B, were isolated from water of ponds of two salterns located in Huelva (Spain). They were Gram-stain-negative, aerobic and slightly curved rods. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the four strains belong to the genus Idiomarina, being related most closely to Idiomarina fontislapidosi F23T (98.4-98.0% sequence similarity), Idiomarina seosinensis CL-SP19T (98.3-98.0%), Idiomarina piscisalsi TPS4-2T (97.9-97.4%), Idiomarina baltica OS145T (97.5-97.4%) and Idiomarina zobellii KMM 231T (97.6-97.0%). The level of similarity with the type species of the genus, Idiomarina abyssalis KMM 227T, was 97.2-96.7%. The novel strains exhibited optimal growth at 5-10% (w/v) total salts, pH 7 and 37 °C. The major fatty acids of strain SN-14T were iso-C15 : 0, iso-C17 : 0, C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and iso-C17 : 1ω9c/C16 : 0 10-methyl. The DNA G+C content range was 47.6-50.8 mol%. The level of DNA-DNA relatedness between strain SN-14T and I. fontislapidosi F23T was 13%, while those between strain SN-14T and the other three new isolates were between 77 and 99%. These data demonstrated that the four isolates constitute a novel species of the genus Idiomarina. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the four strains represent a novel species of the genus Idiomarina, for which the name Idiomarina aquatica sp. nov. is proposed. The type strain is SN-14T ( = CCM 8471T = CECT 8360T = LMG 27613T).

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.

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 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.

La biodiversidad microbiana como fuente de productos de interés biotecnológico / No disponible

Introducción: La diversidad microbiana es una fuente importante de productos de interés biotecnológico, tales como antibióticos, enzimas o Polímeros 11,12. Actualmente la biodiversidad está viéndose seriamente afectada por la contaminación y por la intervención del hombre en la naturaleza. La pérdida de microorganismos no solo alterará los ecosistemas sino que supondrá la desaparición de productos de interés biotecnológico. La biodiversidad microbiana permanece aún inexplorada porque con los métodos clásicos de cultivo microbiano sólo aislamos en el laboratorio entre el 0,1 y el 10% de los microorganismos presentes en determinado ecosistema. En cambio la aplicación de herramientas moleculares permite estudiar la diversidad, estructura y la dinámica de comunidades microbianas de diferentes y complejos ambientes, así como detectar la presencia de microorganismos con interés para la industria farmacéutica y la agricultura, entre otras áreas. Objetivo: Analizar y estudiar la diversidad microbiana mediante técnicas moleculares, tanto en ambientes libres de contaminación como es el Parque natural Rambla Salada (Murcia), como en otros hábitatsc ontaminados por las actividades humanas (suelos agrícolas de Motril, Granada)(AU)

Metodología: El estudio de la diversidad microbiana requirió la puesta a punto de una estrategia molecular basada en la amplificación por PCR del gen ribosomal 16S rRNA, a partir del DNA total extraído directamente de las muestras. Posteriormente, se desarrolló la electroforesis en gel con gradiente desnaturalizante (DGGE)1 para la separación de los fragmentos del gen ribosomal 16S ya amplificados, y finalmente se procedió a la secuenciación y comparación de las secuencias obtenidas a partir de los patrones del DGGE con las existentes en las bases de datos. Por otro lado se desarrolló la técnica de hibridación in situ 7 empleando sondas fluorescentes; estas últimas fueron diseñadas para detectar la presencia de microorganismos pertenecientes a los dominios de procariotas Bacteria y Arquea. Discusión y conclusión: Mediante las herramientas moleculares hemos detectado la presencia de arqueas y bacterias no halófilas, halófilas y halotolerantes tanto en todas las zonas analizadas de Rambla Salada como algunas muestras de los suelos agrícolas de Motril. La diversidad microbiana de los suelos agrícolas de Motril es inferior a la de Rambla Salada. Se ha puesto de manifiesto la existencia de una gran diversidad microbiana en Rambla Salada compuesta fundamentalmente por procariotas pertenecientes a los phila Bacteroidetes, Cianobacteria, Proteobacteria, Firmicutes y Actinobacteria cuyos miembros no se aíslan en el laboratorio mediante las técnicas de cultivo empleadas hasta el momento. La presencia de una mayor diversidad microbiana en Rambla Salada indica que los hábitats hipersalinos son una fuente potencial de productos de interés farmacéutico(AU)

Microbial diversity is an important source of products that have potential biotechnological applications, such as antibiotics, enzymes or polymers [1, 2]. The biodiversity is seriously affected by pollution and human impact on natural environments. In this sense, reduction of biodiversity not only alters the ecosystems but also will entail the disappearance of products having biotechnological interest. Microbial biodiversity is not enough known because we have only be able so far to culture between 0.1 and 10% of the microorganisms that exist in nature. Never the less, molecular ecologytechniques represent an opportunity to study the diversity, structure and dynamics of these uncultured microorganisms, both in diverse and complex environments. They also are useful tools to detect Microorganisms that are of interest to pharmaceutical industry and agriculture, among to other areas(AU)

Therefore, the main objective of this work has been to analyse the microbial diversity in different environments using molecular methods. The habitats studied were an hypersaline soil located at Rambla Salada (Murcia), and some agricultural soils from Motril (Granada). We used PCR/DGGE to investigate the microbial diversity; this method is based on the amplification of partial 16S RNAr geneusing the total DNA extracted directly from the sample. Subsequently denaturant gradient gel electrophoresis (DGGE) [3] is developed to separate the amplified fragments of the 16S RNAr gene. Finally the sequences obtained from the DGGE patterns are compared to those available at the database. On the other hand, we used fluorescence in situ hybridization or FISH [4] to detect and quantify microorganisms be longing to the Domain Bacteria and Archaea. According to our results, non halophilic, halophilic, and halotolerant Archaea and Bacteria were present in all the areas analyzed at Rambla Salada, as well as in some samples of the agricultural soils in Motril. Microbial diversity found in the agricultural soils in Motril was lower than in Rambla Salada. Microbial community at Rambla Salada was composed of Prokaryotes belonging to the phyla Bacteroidetes, Cyanobacteria, Proteobacteria, Firmicutes and Actinobacteria, which cannot not so far isolated using methods based on traditional culture techniques(AU)

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.