Ana del Moral

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).

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.

Transposon mutagenesis in Halomonas eurihalina

We have established a transposon mutagenesis procedure for the moderate halophile Halomonas eurihalina, a bacteria that produces an exopolysaccharide (EPS) of considerable biotechnological interest. We used suicide plasmids pUT and pSUP102 to introduce the transposons mini-Tn5 and Tn1732 into H. eurihalina via Escherichia coli mediated conjugation. Southern hybridization analysis demonstrated that insertions of the transposon mini-Tn5 into H. eurihalina occurred randomly at single sites in the chromosome, whereas Tn1732 insertion also took place at random, but simultaneously, at several sites. Phenotypic analysis revealed that different mutants were generated by using mini-Tn5. The isolation of exopolysaccharide-defective strains is the first stage towards carrying out genetic studies on EPS production by this microorganism.

Megaplasmids in Gram-negative, moderately halophilic bacteria

We have discovered that many Halomonas species harbour large extrachromosomal DNA elements. Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide. We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids. Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements. The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings.

Numerical taxonomy of microorganisms isolated from goat cheese made in Chile.

Abstract118 strains of heterotrophic microorganisms were isolated from goat cheese produced domestically in the IV Region of Northern Chile (Serene, Ovalle, and Illapel) and sold in supermarkets in Valparaíso, Chile. The results of 89 phenotypic tests were numerically analyzed against 17 reference strains, using the simple matching coefficient (SSM). Thirteen phena were found at a 78% similarity level. Five of them (A, B, C, D, and E) were assigned to the family Enterobacteriaceae, phenon F was identified as belonging to the genus Aeromonas and strains of phenon G were assigned to the genus Acinetobacter. The other phena were identified as being members of the genera Bacillus (H, I, and J), Staphylococcus (K), Enterococcus (L), and Micrococcus (M). Approximately 19% of the isolates were Escherichia coli and 27%, Staphylococcus aureus.

Analysis of the Genome of the Moderate Halophile Halomonas eurihalina

We have studied the genomic organization of Halomonas eurihalina, a moderately halophilic bacterium that produces an anionic exopolysaccharide with a potentially wide range of applications in industry. To estimate the genome size of strain F2-7, large restriction fragments of genomic DNA were separated by pulsed-field gel electrophoresis. According to the average size of the resolved restriction fragments, the genome size of H. eurihalina strain F2-7 was estimated to be around 2500 kb. The physical map of the chromosome for the endonuclease SwaI has been constructed. The F2-7 strain has two plasmids, pVE1 and pVE2, and in this study we have isolated three new plasmids, pVE3, pVE4, and pVE5, of 5.3, 16, and 6.5 kb, respectively, from strains H-1, H-217, and H-236 of H. eurihalina. We have characterized these plasmids and constructed their physical maps. Curing experiments produced no evidence for the involvement of these plasmids in the expression of the mucous phenotype.

Chemical mutagenesis of Halomonas eurihalina and selection of exopolysaccharide-deficient variants

The halophilic bacteria Halomonas eurihalina produces an exopolysaccharide (EPS) of potential interest for biotechnological applications. To study the characteristics of this bacteria and others like it, we have developed an efficient chemical mutation technique using hydroxylamine and, in this way, have obtained non-mucoid mutants of its F2-7 strain. We have characterised the phenotypes of the mutants via nutritional, biochemical and physiological tests and by their susceptibility to antimicrobial agents and heavy metals. These mutants will be used as recipient tools in further studies to discover the genetic determinants for the synthesis of EPS.

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.

Taxonomic Study of Exopolysaccharide-producing, Moderately Halophilic Bacteria Isolated from Hypersaline Environments in Morocco

Summary A total of 46 strains of moderately halophilic bacteria were isolated from various solar salterns in Morocco. All the strains had the ability to produce exopolysaccharides. We studied their phenotypes by morphological and physiological observations, biochemical and nutritional testing and antimicrobial susceptibility. The results were subjected to numerical analysis using the Simple Matching (S SM ) coefficient and clustered by the unweighted-pair-group method of association (UPGMA). Two phena were obtained at an 80% similarity level. Representative strains from both phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. Bacteria belonging to phenon A could be included within the genus Halomonas , although they were not closely related to any of its hitherto recognised species. Strains of phenon B were phenotypically related to the moderately halophilic species of Bacillus , but DNA relatedness studies showed that they were not intimately related to them neither to the species of the genus Halobacillus . These results suggest that the exopolysaccharide-producing strains may well constitute new taxa of moderately halophilic bacteria.

Distribution and types of Heterotrophyc Halophilic Flora from Salar de Atacama, chile

Samples were taken at different saline habitats (water, soils and sediments) in the Salar de Atacama (Chile) and later quantitatively analysed using media with a wide range of salt concentrations. Results obtained were nearly similar in all places studied, although counts differed depending on salt concentrations in the media. A total of 361 moderately and 75 extremely halophilic strains were selected and their preliminary taxonomic position was studied.