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.

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Numerical Taxonomy of Moderately Halophilic Gram-negative Rods

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.

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

Five brightly red-pigmented, motile, rod-shaped, extremely halophilic bacteria were isolated from saltern crystallizer ponds in Alicante (two strains) and Mallorca (three strains), Spain. They grew optimally at salt concentrations between 20 and 30% and did not grow below 15% salts. Thus, these isolates are among the most halophilic organisms known within the domain Bacteria. The temperature optimum was 37-47 degrees C. A single, yet to be identified pigment was present, with an absorption maximum at 482 nm and a shoulder at 506-510 nm. The G+C content of the DNA was 66.3-67.7 mol% and, together, they formed a homogeneous genomic group with DNA-DNA similarities above 70%. The 16S rRNA gene sequences were almost identical to sequences recovered earlier from the saltern biomass by amplification of bacterial small-subunit rRNA genes from DNA extracted from the environment. This phylotype, earlier described as 'Candidatus Salinibacter', was shown by fluorescence in situ hybridization to contribute between 5 and 25% of the prokaryote community of the saltern crystallizers. We have therefore succeeded in isolating a bacterium from the natural environment that, although being a major component of the community, was previously known by its phylotype only. Isolation of the organism now allows formal description of a novel genus and species, for which we propose the name Salinibacter ruber gen. nov., sp. nov. The type strain is strain M31T (= DSM 13855T = CECT 5946T).

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Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds.

A Gram-positive, extremely halotolerant bacterium was isolated from the Great Salt Lake, Utah, USA. The strain, designated NNT(= DSM 11805T), was strictly aerobic, rod-shaped, motile by peritrichous flagella and spore-forming. Strain NNTgrew at salinities of 0-20% (w/v) NaCl. A distinctive feature of strain NNTwas its optimal growth in salt-free medium. The polar lipid pattern of strain NNTconsisted of phosphatidyl glycerol, diphosphatidyl glycerol and two phospholipids of unknown structure. The G+C content of its DNA was 38 mol%. The morphological, physiological and, particularly, the 16S rDNA sequence data, showed that strain NNTwas associated with ‘Bacillus group 1’. However, the organisms showing the greatest degree of sequence similarity to strain NNTwere members of the genus Halobacillus and the species Marinococcus albus, Virgibacillus pantothenticus, Bacillus salexigens and Bacillus dipsosauri. On the basis of chemotaxonomic data, strain NNTwas shown to be chemically most similar to B. salexigens and B. dipsosauri, with the greatest degree of similarity being shown to the latter organism. This was consistent with the 16S rDNA sequence data. Members of the genus Halobacillus comprise a chemically distinct group and can easily be distinguished from all other organisms of ‘Bacillus group 1’. On the basis of the 16S rDNA data, chemotaxonomy and the physiology of strain NNT, it is proposed that this organism is a member of a new species, within a new genus, for which the name Gracilibacillus halotolerans is proposed. It is also proposed that B. dipsosauri be transferred to this genus as Gracilibacillus dipsosauri comb. nov. and that B. salexigens be transferred to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Finally, additional data is provided to support the transfer of Bacillus pantothenticus to the genus Virgibacillus, as Virgibacillus pantothenticus Heyndrickx et al. (1998).

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Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov.

A vast and rich body of information has grown up as a result of the world's enthusiasm for 'omics technologies. Finding ways to describe and make available this information that maximise its usefulness has become a major effort across the 'omics world. At the heart of this effort is the Genomic Standards Consortium (GSC), an open-membership organization that drives community-based standardization activities, Here we provide a short history of the GSC, provide an overview of its range of current activities, and make a call for the scientific community to join forces to improve the quality and quantity of contextual information about our public collections of genomes, metagenomes, and marker gene sequences.

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The Genomic Standards Consortium

Various workers attempting to stain the halophilic bacteria which are commonly responsible for the red discoloration of salted hides and salted fish have been unanimous in reporting major difficulties. These organisms are ordinarily grown in media containing 20 per cent or more of NaCl. They are extremely sensitive to changes in osmotic pressure and will undergo plasmoptysis immediately when immersed in distilled water. Consequently, smears for microscopical examination should be made in 20 per cent brine. Then, if a good preparation is to be obtained, a special desalting procedure must be applied before the actual staining of the organisms.

H. P. Dussault

Papers

An improved technique for staining red halophilic bacteria