Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production / El análisis genómico comparativo de cepas mutadas por radiación ultravioleta y de tipo salvaje de Halomonas campaniensis revela diferencias genómicas asociadas con una mayor producción de ectoína

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L−1 (0.65 g g−1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L−1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L−1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.(AU)

Medicago sativa L: mejora y nuevos aspectos de su valor nutritivo y funcional por coinoculación bacteriana / Medicago sativa l: improvement and new approaches of its nutritional and functional value by bacterial co-inoculation

Objetivo: estudiar el efecto de la inoculación con Ensifer meliloti y Halomonas maura sobre el crecimiento y el valor nutricional y funcional de la leguminosa Medicago sativa L., cultivada bajo condiciones de salinidad. Método: las plantas de M. sativa se cultivaron con una solución de mezcla de sales CaSO4 , MgCl, NaCl and NaHCO3 y se coinocularon con su rizobio específico y la bacteria H. maura. Se determinaron los parámetros fisiológicos de las plantas, así como el contenido en nitrógeno y minerales, y se llevó a cabo un proceso de digestibilidad in vitro. Resultados: la salinidad ejerció un efecto negativo sobre las plantas; sin embargo, la coinoculación de las mismas incrementó su productividad, el contenido en nitrógeno, minerales totales, Ca y Mg. Además, los parámetros fisiológicos de potencial hídrico y concentración de leghemoglobina se incrementaron. Tanto la salinidad como la coinoculación de las plantas aumentaron la capacidad antioxidante de la leguminosa en los dializados y retenidos obtenidos tras someter a la planta a un proceso de digestibilidad in vitro. Conclusión: la coinoculación con E. meliloti y H. maura podría mejorar el cultivo de la alfalfa bajo condiciones específicas de salinidad, aumentando su composición nutricional y funcional, pudiendo considerarse en la formulación de suplementos nutricionales para el consumo humano (AU)

Objective: to study the effect of co-inoculation with Ensifer meliloti and Halomonas maura of the leguminous Medicago sativa L., on growth, nutritional and functional value, grown under salinity conditions. Methods: plants of M. sativa were grown in a solution with a mixture of salts (CaSO4 , MgCl, NaCl and NaHCO3 ) and were co-inoculated with its specific rhizobium and the halophilic moderated bacterium H. maura. Different physiologic parameters were determined, as well as, nitrogen and minerals content. Furthermore, an assay of in vitro digestibility was carried out. Results: salinity had a negative effect on the plants; however, co-inoculation increased yield, nitrogen content, total minerals, Ca and Mg. Moreover, physiologic parameters as water potential and leghemoglobin content in fresh nodules were higher compared to those of plants inoculated only with E. meliloti. Both, salinity and bacterial treatment with E. meliloti and H. maura increased the antioxidant capacity of the legume, in dialyzates and retentates collected after an in vitro digestibility assay. Conclusion: co-inoculation of plants with E. meliloti and H. maura could improve the alfalfa yield under specific salinity conditions, increasing the nutritional and functional value of the plants. M. sativa could be considered in the formulations of nutritional supplements for the human diet (AU)

Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulating Halomonas venusta

Biofilm development is characterized by distinct stages of initial attachment, microcolony formation and maturation (sessile cells), and final detachment (dispersal of new, planktonic cells). In this work we examined the influence of polyhydroxyalkanoate (PHA) accumulation on bacterial surface properties and biofilm formation on polystyrene in detached vs. planktonic cells of an environmental strain isolated from microbial mats, Halomonas venusta MAT28. This strain was cultured either in an artificial biofilm in which the cells were immobilized on alginate beads (sessile) or as free-swimming (planktonic) cells. For the two modes of growth, conditions allowing or preventing PHA accumulation were established. Cells detached from alginate beads and their planktonic counterparts were used to study cell surface properties and cellular adhesion on polystyrene. Detached cells showed a slightly higher affinity than planktonic cells for chloroform (Lewis-acid) and a greater hydrophobicity (affinity for hexadecane and hexane). Those surface characteristics of the detached cells may explain their better adhesion on polystyrene compared to planktonic cells. Adhesion to polystyrene was not significantly different between H. venusta cells that had accumulated PHA vs. those that did not. These observations suggest that the surface properties of detached cells clearly differ from those of planktonic cells and that for at least the first 48 h after detachment from alginate beads H. venusta retained the capacity of sessile cells to adhere to polystyrene and to form a biofilm (AU)

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Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artificial biofilms of alginate beads

Microbial mats are complex but stable, multi-layered and multi-functional biofilms, which are the most frequent bacterial formations in nature. The functional strategies and physiological versatility of the bacterial populations growing in microbial mats allow bacteria to resist changing conditions within their environment. One of these strategies is the accumulation of carbon- and energy-rich polymers that permit the recovery of metabolic activities when favorable conditions are restored. In the present study, we systematically screened microbial mats for bacteria able to accumulate large amounts of the ester carbon polymers polyhydroxyalkanoates (PHA). Several of these strains were isolated from Ebro Delta microbial mats and their ability to accumulate PHA up to 40-60% of their dry weight was confirmed. According to two identification approaches (16S rRNA and rpoD genes), these strains were identified as Halomonas alkaliphila (MAT-7, -13, -16), H. neptunia (MAT-17), and H. venusta (MAT-28). To determine the mode of growth yielding maximum PHA accumulation, these three different species were cultured in an artificial biofilm in which the cells were immobilized on alginate beads. PHA accumulation by cells that had detached from the biofilm was compared with that of their planktonic counterparts. Experiments in different culture media showed that PHA accumulation, measured as the relative fluorescence intensity after 48 h of incubation at 30 degrees C, was higher in immobilized than in planktonic cells, with the exception of cells growing in 5% NaCl, in which PHA accumulation was drastically lower in both. Therefore, for obtaining high PHA concentrations, the use of immobilized cells may be a good alternative to the PHA accumulation by bacteria growing in the classical, planktonic mode. From the ecological point of view, increased PHA accumulation in detached cells from biofilms would be a natural strategy to improve bacterial dispersion capacity and, consequently, to increase survival in stressed environments (AU)

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Aislamiento de cepas del género Halomonas con interés biotecnológico en Rambla Salada (Murcia) / Isolaton of strains of the genus Halomonas with biotechnologycal interest from Rambla Salada (Murcia)

El género Halomonas es uno de los taxones bacterianos más representativos dentro de las bacterias halófilas moderadas. Se encuentra incluido en el Phyllum Proteobacteria, clase Gamma-Proteobacteria, familia Halomonadaceae1. Son en su mayoría de bacterias halófilas moderadas, con un requerimiento de NaCl para su óptimo crecimiento que oscila entre un 3-15% (p/v). Las especies de esta familia han demostrado ser un grupo de microorganismos extremófilos con un gran potencial biotecnológico, por la producción de solutos compatibles que les confiere capacidad para estabilizar y proteger enzimas así como aplicaciones en tecnología enzimática, en la industria cosmética y dermofarmacia, medicina y agricultura. Estas especies son capaces de producir también compuestos extracelulares como exopolisacáridos y enzimas extracelulares. Los primeros tienen gran interés industrial debido a sus propiedades viscosizantes, estabilizantes, emulgentes y formadoras de geles. Nuestro grupo de investigación a lo largo de estos años ha caracterizado una decena de estos polímeros destacando los de H. eurihalina y H. maura por sus propiedades viscosizantes y emulgentes 2,3 y los de H. stenophila por sus propiedades antitumorales 4. En cuanto a las enzimas extracelulares, tales como lipasas y amilasas, se mantienen estables en un amplio rango de concentraciones salinas y proporcionan así nuevas posibilidades en los procesos biocatalíticos. Otras aplicaciones biotecnológicas son la producción de polihidroxialcanoatos así como la degradación de compuestos tóxicos. Debido al gran interés biotecnológico de las bacterias halófilas moderadas, nuestro grupo de investigación lleva a cabo un estudio de biodiversidad de microorganismos halófilos en Rambla Salada (Murcia) con el fin de aislar nuevas especies de Halomonas productoras de compuestos de aplicación industrial y/o biotecnológica(AU)

Se han realizado cuatro muestreos durante dos años en Rambla Salada (Murcia, España). Las muestras han sido procesadas mediante las técnicas de microbiología clásicas y las bacterias aisladas han sido caracterizadas fenotípica y filogenéticamente. Las cepas pertenecientes al género Halomonas se sembraron en los medios adecuados para estudiar cada una de las funciones biológicas, y se seleccionaron aquellas cepas que dieron una actividad positiva. Se han aislado 164 cepas del género Halomonas, algunas de ellas con actividad lipasa, proteinasa y/odexosirribonucleasa y casi todas son productoras de exopolisacaridos. La presencia de estas cepas conactividad positiva para determinadas funciones biológicas con interés industrial y biotecnológico ponen de manifiesto la necesidad de seguir investigando en este sentido(AU)

The genus Halomonas is one of the most representative taxa amongst the moderately halophilicbacteria. This genus is included into the family Halomonadaceae, class Gamma-Proteobacteria, Phyllum Proteobacteria1. It requires NaCl concentrations 3-15% w/v for optimum growth. Halomonas species have aroused interest amongst biotechnologists because of their ability to accumulate organic compatible solutes which provide stability and protection of enzymes and other molecules and have interest in enzymatic technology, cosmetic, medicine and agriculture. They also produce extracellular compounds, such as exoenzymes and exopolysaccharides (EPS). The extracellular enzymes, such as lipase and amylase, are stable within a wide salt concentration range; this property is important in biocatalytic processes. Other biotechnological applications of the Halomonas species are the production of polyhydroxyalkanoates and the degradation of aromatic compounds. The halophilic EPS are commercially valuable products because of their biotechnology applications as viscosifying, stabilizers and gelling agents. Our research group has characterized to date tenexopolysaccharides-producing halophilic species. Two of them, H. eurihalina and H. maura produce polymers with viscosifying and emulsifying properties 2,3 and H. stenophila synthesizes and EPS with antitumoral properties 4. Isolate new species of the genus Halomonas with industrial and/or medical applications from Rambla Salada (Murcia, Spain)(AU)

Samples of saline soils, sediments and waters were taken periodically from Rambla Salada (Murcia, Spain) along two years. The samples were processed by classical methods and the isolated strains were identified by mean of phenotypic and phylogenetic techniques. The species belonging to the genus Halomonas were analyzed in order to find compounds of biotechnological interest. We have isolated 164 strains of the genus Halomonas. Some of them have lipases protease and/ordeoxyribonuclease activities and most of them are exopolysaccharide-producing bacteria. The results presented in this work show that extreme environments, such as Rambla Salada, constitute anunexplorable resource of discovering new bacteria and biomolecules(AU)