Antonio Ventosa

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.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Proposed Minimal Standards for Description of New Taxa in the Order Halobacteriales

In accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, which calls for the development of minimal standards for describing new species, we propose minimal standards for description of new taxa in the order Halobacteriales. The minimal standards include information on the following characteristics: cell morphology; motility; pigmentation; the requirement for salt to prevent cell lysis; optimum NaCl and MgCl2 concentrations for growth and range of salt concentrations enabling growth; temperature and pH ranges for growth; anaerobic growth in the presence of nitrate or arginine; acid production from a range of carbohydrates; ability to grow on single carbon sources; catalase and oxidase tests; hydrolysis of starch, casein, and Tween 80; sensitivity to different antibiotics; and polar lipids. The placement of a new taxon should be consistent with phylogeny, which is usually based on 16S rRNA nucleotide sequence information, and with DNA-DNA hybridization data in the case of descriptions of new species. This proposal has been endorsed by the members of the Subcommittee on the Taxonomy of Halobacteriaceae of the International Committee on Systematic Bacteriology.

Classification of Non-alkaliphilic Halobacteria Based on Numerical Taxonomy and Polar Lipid Composition, and Description of Haloarcula gen. nov. and Haloferax gen. nov.

Summary A large number of halobacteria have been isolated from different hypersaline environments at different geographical sites and studied together with type culture collection strains with respect to several phenotypic features. Numerical processing of the data resulted in thirteen phenons. A representative of each was studied to determine its polar lipid composition. Our results show the existence of at least three groups with the taxonomic level of genus. Results obtained by other authors with regard to the genotypic relationships among halobacteria support this. Besides the existing genus Halobacterium we propose the two new genera Haloarcula and Haloferax for the nomenclatural accomodation of the three groups.

Biotechnological applications and potentialities of halophilic microorganisms.

Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represented, but not exclusively, by the halobacteria (extremely halophilic aerobic Archaea), the moderate halophiles (Bacteria and some methanogens) and several eukaryotic algae. These extremophilic microorganisms are already used for some biotechnological processes, for example halobacteria are used for the production of bacteriorhodopsin, and the alga Dunaliella is used in the commercial production of β-carotene. Several other present or potential applications of halophiles are reviewed, including the production of polymers (polyhydroxyalcanoates and polysaccharides), enzymes, and compatible solutes, and the use of these extremophiles in enhanced oil recovery, cancer detection, drug screening and the biodegradation of residues and toxic compounds.

Halobacillus gen. nov., with Descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and Transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov.

Two moderately halophilic, gram-positive, heterotrophic bacterial strains were isolated from hypersaline sediments of the Great Salt Lake in Utah. These two strains, designated SL-4T (T = type strain) and SL-5T, were motile, spore-forming, strictly aerobic rods which contained peptidoglycan of the Orn-D-Asp type in their vegetative cell walls. The guanine-plus-cytosine contents of the DNAs of strains SL-4T and SL-5T were 42 and 43 mol%, respectively. A detailed investigation of the phenotypic and phylogenetic characteristics of these organisms revealed that each isolate represents a new species that is closely related to Sporosarcina halophila, a moderately halophilic, spore-forming coccus. Phylogenetic data indicate that there is only a distant relationship between Sporosarcina halophila and Sporosarcina ureae, the type species of the genus Sporosarcina. The sequences of the 16S rRNA genes of strain SL-4T and Salinicoccus roseus DSM 5351 were determined. We propose that a new genus, Halobacillus, should be created; this genus includes Halobacillus halophilus (formerly Sporosarcina halophila) as the type species, as well as Halobacillus litoralis DSM 10405T (= SL-4T) and Halobacillus trueperi DSM 10404T (= SL-5T).

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).

Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern

A multi-pond saltern that creates a gradient of salt concentrations has been studied with respect to some characteristics of the resulting environments and their microbial populations. The increase in salt concentration was correlated with increase in diurnal temperature and biomass present and with decrease in oxygen concentrations. Many types of organisms below 15% (w/v) total salts, were found, many of them normal inhabitants of seawater and even freshwater. Most organisms over 15% salts were halophilic. The salt concentrations comprised two ranges, each characterized by different microbial populations. First, between 15 and 30% salts, the populations ofDunaliella increased, reaching large numbers; moderately halophilic eubacteria and some fast-growing halobacteria predominated as heterotrophic microorganisms and, among the first, thePseudomonas-Alteromonas-Alcaligenes group andVibrio were the more abundant taxonomic groups; and gram-positive cocci appeared mainly over 25% salts. Phototrophic bacteria, both oxygenic and anoxygenic, were also found in this range, and among the anoxygenic type,Chromatium species andRodospirillum salexigens were probably predominant. Second, over 30% salts the diversity decreased greatly, all organisms found at the lower salt concentrations disappeared, and instead large populations of halobacteria developed. Over 50% salts, only three species of halobacteria were found.

New Abundant Microbial Groups in Aquatic Hypersaline Environments

We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.

Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran

Screening of bacteria from different areas of Howz Soltan playa, a hypersaline lake in the central desert zone of Iran, led to the isolation of 231 moderately halophilic bacteria, which were able to grow optimally in media with 5–15% of salt, and 49 extremely halophilic microorganisms that required 20–25% of salt for optimal growth. These isolates produced a great variety of extracellular hydrolytic enzymes. A total of 195, 177, 100, 95, 92, 68, 65, 33, and 28 strains produced lipases, amylases, proteases, inulinases, xylanases, cellulases, pullulanases, DNases, and pectinases, respectively. In comparison with gram-negative bacteria, the gram-positive halophilic rods, showed more hydrolytic activities. Several combined activities were showed by some of these isolates. One strain presented 9 hydrolytic activities, 4 strains presented 8 hydrolytic activities, 10 strains presented 7 hydrolytic activities and 29 strains presented 6 hydrolytic activities. No halophilic isolate without hydrolytic activity has been found in this study. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the halophilic strains were identified as members of the genera: Salicola, Halovibrio, Halomonas, Oceanobacillus, Thalassobacillus, Halobacillus, Virgibacillus, Gracilibacillus, Salinicoccus, and Piscibacillus. Most lipase and DNase producers were members of the genera Gracilibacillus and Halomonas, respectively, whereas most of the isolates able to produce hydrolytic enzymes such as amylase, protease, cellulose (CMCase) and inulinase, belonged to gram-positive genera, like Gracilibacillus, Thalassobacillus, Virgibacillus, and Halobacillus.