Proposed minimal standards for description of new taxa of the class Halobacteria.

Halophilic archaea of the class Halobacteria are the most salt-requiring prokaryotes within the domain Archaea. In 1997, minimal standards for the description of new taxa in the order Halobacteriales were proposed. From then on, the taxonomy of the class Halobacteria provides an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods were implemented. The last decades have witnessed a rapid expansion of the number of described taxa within the class Halobacteria coinciding with the era of genome sequencing development. The current members of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteria propose these revisions to the recommended minimal standards and encourage the use of advanced technologies in the taxonomic description of members of the Halobacteria. Most previously required and some recommended minimal standards for the description of new taxa in the class Halobacteria were retained in the present revision, but changes have been proposed in line with the new methodologies. In addition to the 16S rRNA gene, the rpoB' gene is an important molecular marker for the identification of members of the Halobacteria. Phylogenomic analysis based on concatenated conserved, single-copy marker genes is required to infer the taxonomic status of new taxa. The overall genome relatedness indexes have proven to be determinative in the classification of the taxa within the class Halobacteria. Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values should be calculated for rigorous comparison among close relatives.

Halorubrum hochsteinianum sp. nov., an ancient haloarchaeon from a natural experiment.

A single extremely halophilic strain was isolated from salt brine produced when a fresh water lake flooded a large salt mine located beneath the lake. The water that entered this mine contained less than 0.34 M NaCl, but over time, this sealed brine became saturated by Cenozoic age salt (121-125 million-year BCE). The isolated strain requires at least 1.7 M NaCl for survival and grows optimally in 3.1 M NaCl. Therefore, it could not have survived or been present in the waters that flooded this salt mine. The strain grows at a pH range from 6.5 to 9.0 and has a wide tolerance to temperatures from 25 ℃ to at least 60 ℃. The comparison of 16S rRNA and rpoB' genes revealed that strain 1-13-28T is related to Halorubrum tebenquichense DSM 14210T showing 98.6% and 98.1% similarities, respectively. Phylogenetic analyses based on 16S rRNA, rpoB' genes and 122 concatenated archaeal genes show that the strain 1-13-28T consistently forms a cluster with Halorubrum tebenquichense of the genus Halorubrum. Strain 1-13-28T contained sulfated mannosyl glucosyl diether, and the polar lipid profile was identical to those of most Halorubrum species. Based on the overall combination of physiological, phylogenetic, polar lipids and phylogenomic characteristics, strain 1-13-28T (= ATCC 700083T = CGMCC 1.62627T) represents a newly identified species within the genus Halorubrum for which the name Halorubrum hochsteinianum is proposed.

Virgibacillus salarius sp. nov., a halophilic bacterium isolated from a Saharan salt lake.

A Gram-positive, endospore-forming, rod-shaped and moderately halophilic bacterium was isolated from a salt-crust sample collected from Gharsa salt lake (Chott el Gharsa), Tunisia. The newly isolated bacterium, designated SA-Vb1(T), was identified based on polyphasic taxonomy including genotypic, phenotypic and chemotaxonomic characterization. Strain SA-Vb1(T) was closely related to the type strains of Virgibacillus marismortui and Virgibacillus olivae, with 16S rRNA gene sequence similarities of 99.7 and 99.4 %, respectively. However, strain SA-Vb1(T) was distinguished from these two type strains on the basis of phenotypic characteristics and DNA-DNA relatedness (29.4 and 5.1 %, respectively). The genetic relationship between strain SA-Vb1(T) and Virgibacillus pantothenticus IAM 11061(T) (the type strain of the type species) and other type strains of the genus was 96-98 % based on 16S rRNA gene sequence similarity and 18.3-22.3 % based on DNA-DNA hybridization. Biochemical analysis resulted in determination of major fatty acids iso-C(15 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0) (33.3, 29.2 and 9.8 %, respectively); phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine were the main polar lipids and MK-7 was the predominant menaquinone ( approximately 100 %). The distinct characteristics demonstrated by strain SA-Vb1(T) represent properties of a novel species of the genus Virgibacillus, for which the name Virgibacillus salarius sp. nov. is proposed. The type strain is SA-Vb1(T) (=JCM 12946(T) =DSM 18441(T)).

Recommended minimal standards for describing new taxa of the family Halomonadaceae.

Following Recommendation 30b of the Bacteriological Code (1990 Revision), a proposal of minimal standards for describing new taxa within the family Halomonadaceae is presented. An effort has been made to evaluate as many different approaches as possible, not only the most conventional ones, to ensure that a rich polyphasic characterization is given. Comments are given on the advantages of each particular technique. The minimal standards are considered as guidelines for authors to prepare descriptions of novel taxa. The proposals presented here have been endorsed by the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halomonadaceae.

Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives.

The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA-DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate.

A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%-93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2-9-1(T) (= ATCC BAA-75 and JCM 11222) as written in the formal description.