Inmaculada Llamas

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

Four moderately halophilic, exopolysaccharide-producing bacterial strains isolated from soil samples collected from a saltern at Asilah (Morocco) are reported. These four strains were initially considered to belong to the genus Halomonas. Their DNA G+C contents varied between 62.2 and 64.1 mol %. DNA-DNA hybridization revealed a considerable degree of DNA-DNA similarity amongst all four strains (75.5-80.8%). Nevertheless, similarity with the reference strains of phylogenetically close relatives was lower than 40%. 16S rRNA gene sequences were compared with those of other species of Halomonas and other gram-negative bacteria and they were sufficiently distinct phylogenetically from other recognized Halomonas species to warrant their designation as a novel species. The name Halomonas maura sp. nov. is therefore proposed, with strain S-31T (= CECT 5298T= DSM 13445T) as the type strain. The fatty acid composition of strain S-31T revealed the presence of 18:1omega7c, 16:1omega7c/2-OH i15:0 and 16:0 as the major components. Growth rate analysis showed that strain S-31T had specific cationic requirements for Na+ and Mg2+.

Characterization of exopolysaccharides produced by 19 halophilic strains of the species Halomonas eurihalina

The formation, chemical composition and rheological properties of the exopolysaccharides (EPS) produced by 19 strains belonging to Halomonas eurihalina have been compared in two different culture media. Our aim was to screen several strains isolated from saline soils to select those producing maximum EPS yield and good rheological properties. We found that MY medium was best for the production of EPS in all the strains studied. Maximum EPS production was 1.6 g l 1 with strain H212 grown in this medium. The pattern of the chemical composition of the polysaccharides was affected by the strain in question and by the culture medium. All EPS studied had an unusually high sulphate content. Furthermore, the exopolymer from strain H96 contained significant amounts of uronic acid. EPS from strain H96, cultivated in defined NH medium, behaved in an interesting way rheologically; when the pH of the polymer solution was decreased to 3.0 a gel with a viscosity of 30 000 cP formed.

Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones.

ABSTRACT Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as N-acyl homoserine lactones (AHLs). The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the Agrobacterium tumefaciens and Chromobacterium violaceum quorum-sensing systems. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti is rapidly becoming a model organism for the study of quorum sensing. This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa. The Sin system is distinguished among them for the production of long-chain AHLs, including C18-HL, the longest AHL reported so far. In this work, we show that construction of a sinI::lacZ transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity. Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity. We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as Paracoccus denitrificans and Rhodobacter capsulatus. This S. meliloti indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems.

Moderately Halophilic, Exopolysaccharide-Producing Bacteria

Moderate halophiles include a wide array of microorganisms, taxonomically and physiologically distributed among many groups within the Bacteria domain and some groups of the Archaea. Their common characteristic is that they grow best at NaCl concentrations between 0.5 and 2.5 M (Kushner and Kamekura 1988), although they can be found in quite a diverse range of hypersaline habitats (Horikoshi and Grant 1998; Oren 1999).

Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.

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, M8T, 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 M8T 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 × 106 and another of 1.5 × 104 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 maura is a physiologically versatile bacterium of both ecological and biotechnological interest

Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF’s) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.

Characterization of the Exopolysaccharide Produced by Salipiger mucosus A3T, 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.

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C4-HL), N-hexanoyl homoserine lactone (C6-HL), N-octanoyl homoserine lactone (C8-HL) and N-dodecanoyl homoserine lactone (C12-HL). This study suggests that quorum sensing may also play an important role in extreme environments.

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.