Emilia Quesada

Numerical taxonomy of moderately halophilic Gram-negative rods

Summary: A study was made of 516 randomly selected isolates of moderately halophilic bacteria from solar salterns showing salinities between 8.8 and 40.0% (w/v) total salts, located in S.E. Spain. After purification, many cytological, physiological, biochemical, nutritional and antibiotic sensitivity characters were determined for 106 selected saltern isolates and two reference strains. Data were coded and analysed by numerical techniques using the Jaccard coefficient (S j), and clusters of strains were obtained by average linkage (UPGMA) analysis. Nine major phenons were found at the 72.5% similarity level. The properties of each phenon are given, their taxonomic affinities are discussed, and typical reference strains are suggested. Almost all the strains were related to genera known to contain marine species. A large number of the strains could be tentatively assigned to the genus Vibrio, suggesting that this may be an abundant taxon of moderately halophilic Gram-negative rods in solar salterns.

A detailed phenotypic characterisation of the type strains of Halomonas species

We have made a detailed phenotypic characterisation of the type strains of 21 species within the genus Halomonas and have also studied any possible intraspecific variation of strains within H. eurihalina, H. halophila, H. maura and H. salina. We used 234 morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests. Nutritional assays were carried out using both classical and miniaturized (BIOLOG system) identification methods. Two different numerical analyses were made using the TAXAN program; the first included the differential data from all the tests carried out whilst the second used only the 57 tests with the highest diagnostic scores (> or = 0.5). The results of both analyses were quite similar and demonstrated the phenotypic heterogeneity of the Halomonas species in question. At a 62% similarity level the type species were grouped into three phena, the main difference between them being the capacity of those included within phenon A (H. aquamarina, H. meridiana, H. cupida, H. pantelleriensis and H. halmophila) to produce acids from sugars. The species grouped in phenon C (H. campisalis, H. desiderata and H. subglasciescola) used fewer organic substrates than the others. The remaining strains were included in phenon B. H. marisflavi was clearly distinct and thus was not included in any of the three phena. High phenotypic similarity (more than 88%) was found between Halomonas campisalis and Halomonas desiderata. The results of our work should allow researchers to minimise the tests required to arrive at a reliable phenotypic characterisation of Halomonas isolates and to select those of most use to differentiate Halomonas species from each other.

Deleya halophila, a New Species of Moderately Halophilic Bacteria

Deleya halophila sp. nov., which was isolated from hypersaline soils, is proposed. Each of 38 isolates, together with 8 reference strains, was examined in 97 phenotypic tests, and the data were analyzed by numerical taxonomy procedures. The 38 isolates formed a distinct group at a 76% similarity value, as determined by an analysis performed with the simple matching coefficient and unweighted average linkage clustering. Characteristically, the isolates were aerobic, gram-negative rods that were motile by one to eight peritrichous flagella. They grew optimally in the presence of 7.5% (wt/vol) marine salts. The distinguishing features of the new species are its salt requirement, biochemical features, and nutritional versatility. The guanine-plus-cytosine content of the deoxyribonucleic acid is 66.7 mol%. The type strain of this species is strain F5–7 (= CCM 3662).

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+.

Halomonas cerina sp. nov., a moderately halophilic, denitrifying, exopolysaccharide-producing bacterium.

Three bacterial strains were isolated from different saline soils in Spain. The novel strains were moderately halophilic, exopolysaccharide-producing, Gram-negative, non-motile rods. The strains required NaCl and grew best with 7.5-10 % (w/v) NaCl in the medium. They formed wax-coloured colonies, were oxidase-positive and showed respiratory metabolism, using oxygen, nitrate and nitrite as terminal electron acceptors. The novel strains were able to denitrify and did not produce acid from sugars. The DNA G+C contents varied between 62.7 and 66.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and sequence signatures of this gene showed that all three novel isolates belonged to the genus Halomonas in the class Gammaproteobacteria and formed an independent phylogenetic line. The most phylogenetically related species were Halomonas alimentaria, Halomonas campaniensis, Halomonas gudaonensis and Halomonas ventosae, with which the novel strains showed 16S rRNA gene sequence similarity values of between 96.3 and 95.2 %. The principal fatty acids of the novel strains were 16 : 0, 18 : 1 omega 7c, 16 : 1 omega 7c and 19 : 0 cyclo omega 8c. The predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q-9). The name Halomonas cerina sp. nov. is proposed for these isolates. The type strain is SP4T (=CECT 7282T=LMG 24145T).

Numerical Taxonomy of Moderately Halophilic Gram-negative Bacteria from Hypersaline Soils

SUMMARY: A total of 132 moderately halophilic bacteria were isolated from hypersaline soils with a Cl- content between 2.36 and 12.72% (w/v) located near Alicante (S.E. Spain) and examined for 98 phenotypic characteristics including their response to cytological, physiological, biochemical and nutritional tests. They were submitted to a numerical analysis together with six reference strains using both simple matching (SSM ) and Jaccard (SJ ) coefficients, and cluster analysis was carried out by the unweighted pair group method of association (UPGMA), single linkage and complete linkage. With the SJ coefficient and UPGMA clustering, eight phenons were obtained at the 65% similarity level. From each phenon representative strains were chosen for the determination of DNA base composition and for electron microscopy. Bacteria belonging to phenons D, E, and F were assigned to the genus Alcaligenes. Phenon G included 27 strains assigned to Acinetobacter, but the high G + C composition (58.9 mol %) of a representative strain of this phenon suggests that it may represent a new taxon. Phenons A, B, and C were designated Flavobacterium and phenon H was Pseudomonas. The bacteria found in these environments are not related to those from hypersaline waters or normal soils.

Volcaniella eurihalina gen. nov., sp. nov., a moderately halophilic nonmotile gram-negative rod

A comparison of 16 gram-negative moderately halophilic aerobic rod-shaped bacteria with other halophilic and nonhalophilic gram-negative bacteria supported the establishment of Volcaniella eurihalina gen. nov., sp. nov. This comparison included phenotypic properties, salt requirements, and guanine-plus-cytosine contents of the DNAs, as well as DNA-DNA homology studies. The distinguishing features of this new bacterial genus are as follows: The organisms are nonmotile short rods that are oxidase negative; they are aerobic with a strictly respiratory type of metabolism; they are moderate halophiles, optimal growth occurs at a total salt concentration of 7.5% (wt/vol), and they exhibit a strongly euryhaline character; and they have a specific requirement for Na+ ions (sodium can be supplied as NaCl, Na2SO4, or NaBr). The minimum NaCl concentration required is 1.5% (wt/vol). The guanine-plus-cytosine content of the DNA is 59.1 to 65.7 mol%. This organism was isolated from hypersaline habitats, including saline soils and salt ponds, and from seawater. The type strain is strain F9-6 (= ATCC 49336).

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.

Exopolysaccharide production byVolcaniella eurihalina

The application of microbial extracellular polymers began in the 1960s, and since then there has been a remarkable increase in their commercial use. They are used in the food, textile, pharmaceutical, agricultural, paint and petroleum industries.Volcaniella eurihalina, a moderately halophilic eubacterium, produces an extracellular polysaccharide whose physical and chemical properties could be of interest for various industrial applications. The aim of this investigation is to analyze the different environmental parameters which influence the production of polysaccharide, and to study its chemical composition and the rheological properties of its solutions.

Effect of cations, pH and sulfate content on the viscosity and emulsifying activity of the Halomonas eurihalina exopolysaccharide

The effects of monovalent and divalent cations on the rheological behavior of Halomonas eurihalina exopolysaccharide (EPS) were studied. Sodium, potassium, magnesium and calcium were added and the relative abilities to increase viscosity were as follows: KCl > NaCl > MgCl2 > CaCl2. The highest viscosity value was measured in acidic 10−4 M KCl, in which a gel formed. A loss of sulfate content seemed to correlate with the increase of viscosity. H. eurihalina produced EPS in all growth media. Addition of hydrophobic substrates to culture media produced changes in chemical composition and emulsifying activity of the EPS. Xylene was the most effectively emulsified substance and the EPS produced on tetradecane and on corn oil the most active emulsifier.