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.

Cultivation of halophilic archaea (class Halobacteria) from thalassohaline and athalassohaline environments.

As a group, the halophilic archaea (class Halobacteria) are the most salt-requiring and salt-resistant microorganisms within the domain Archaea. Halophilic archaea flourish in thalassohaline and athalassohaline environments and require over 100-150 g/L NaCl for growth and structural stability. Natural hypersaline environments vary in salt concentration, chemical composition and pH, and occur in climates ranging from tropical to polar and even under-sea. Accordingly, their resident haloarchaeal species vary enormously, as do their individual population compositions and community structures. These diverse halophilic archaeal strains are precious resources for theoretical and applied research but assessing their taxonomic and metabolic novelty and diversity in natural environments has been technically difficult up until recently. Environmental DNA-based high-throughput sequencing technology has now matured sufficiently to allow inexpensive recovery of massive amounts of sequence data, revealing the distribution and community composition of halophilic archaea in different hypersaline environments. While cultivation of haloarchaea is slow and tedious, and only recovers a fraction of the natural diversity, it is the conventional means of describing new species, and provides strains for detailed study. As of the end of May 2020, the class Halobacteria contains 71 genera and 275 species, 49.8% of which were first isolated from the marine salt environment and 50.2% from the inland salt environment, indicating that both thalassohaline and athalassohaline environments contain diverse halophilic archaea. However, there remain taxa that have not yet been isolated in pure culture, such as the nanohaloarchaea, which are widespread in the salt environment and may be one of the hot spots in the field of halophilic archaea research in the future. In this review, we focus on the cultivation strategies that have been used to isolate extremely halophilic archaea and point out some of the pitfalls and challenges. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-020-00087-3.

The Genome Sequence of the Halobacterium salinarum Type Strain Is Closely Related to That of Laboratory Strains NRC-1 and R1.

High-coverage long-read sequencing of the Halobacterium salinarum type strain (91-R6) revealed a 2.17-Mb chromosome and two large plasmids (148 and 102 kb). Population heterogeneity and long repeats were observed. Strain 91-R6 and laboratory strain R1 showed 99.63% sequence identity in common chromosomal regions and only 38 strain-specific segments. This information resolves the previously uncertain relationship between type and laboratory strains.

NaCl-saturated brines are thermodynamically moderate, rather than extreme, microbial habitats.

NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.

Genome Sequence of an Australian Monophasic Salmonella enterica subsp. enterica Typhimurium Isolate (TW-Stm6) Carrying a Large Plasmid with Multiple Antimicrobial Resistance Genes.

We report the genome sequence of a monophasic Salmonella enterica subsp. enterica Typhimurium strain (TW-Stm6) isolated in Australia that is similar to epidemic multidrug-resistant strains from Europe and elsewhere. This strain carries additional antibiotic and heavy-metal resistance genes on a large (275-kb) IncHI2 plasmid.

Taxonomic study of the genera Halogeometricum and Halosarcina: transfer of Halosarcina limi and Halosarcina pallida to the genus Halogeometricum as Halogeometricum limi comb. nov. and Halogeometricum pallidum comb. nov., respectively.

Members of the haloarchaeal genera Halosarcina and Halogeometricum (family Halobacteriaceae) are closely related to each other and show 96.6-98 % 16S rRNA gene sequence similarity. This is higher than the accepted threshold value (95 %) to separate two genera, and a taxonomic study using a polyphasic approach of all four members of the two genera was conducted to clarify their relationships. Polar lipid profiles indicated that Halogeometricum rufum RO1-4(T), Halosarcina pallida BZ256(T) and Halosarcina limi RO1-6(T) are related more to each other than to Halogeometricum borinquense CGMCC 1.6168(T). Phylogenetic analyses using the sequences of three different genes (16S rRNA gene, rpoB' and EF-2) strongly supported the monophyly of these four species, showing that they formed a distinct clade, separate from the related genera Halopelagius, Halobellus, Haloquadratum, Haloferax and Halogranum. The results indicate that the four species should be assigned to the same genus, and it is proposed that Halosarcina pallida and Halosarcina limi be transferred to the genus Halogeometricum as Halogeometricum pallidum comb. nov. (type strain, BZ256(T) = KCTC 4017(T) = JCM 14848(T)) and Halogeometricum limi comb. nov. (type strain, RO1-6(T) = CGMCC 1.8711(T) = JCM 16054(T)).

Genome of the haloarchaeon Natronomonas moolapensis, a neutrophilic member of a previously haloalkaliphilic genus.

The genus Natronomonas contains two species, one haloalkaliphile (N. pharaonis) and one neutrophile (N. moolapensis). Here, we report the genome sequence of N. moolapensis strain 8.8.11. The overall genome properties are similar for the two species. Only the neutrophile contains bacteriorhodopsin and a membrane glycolipid.

Halohasta litorea gen. nov. sp. nov., and Halohasta litchfieldiae sp. nov., isolated from the Daliang aquaculture farm, China and from Deep Lake, Antarctica, respectively.

Two halophilic archaeal strains, R30(T) and tADL(T), were isolated from an aquaculture farm in Dailing, China, and from Deep Lake, Antarctica, respectively. Both have rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require >120 g/l NaCl and 48-67 g/l MgCl(2) for growth but differ in their optimum growth temperatures (30 °C, tADL(T) vs. 40 °C, R30(T)). The major polar lipids were typical for members of the Archaea but also included a major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). The 16S rRNA gene sequences of the two strains are 97.4 % identical, show most similarity to genes of the family Halobacteriaceae, and cluster together as a distinct clade in phylogenetic tree reconstructions. The rpoB' gene similarity between strains R30(T) and tADL(T) is 92.9 % and less to other halobacteria. Their DNA G + C contents are 62.4-62.9 mol % but DNA-DNA hybridization gives a relatedness of only 44 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe two new species of a novel genus, represented by strain R30(T) (= CGMCC 1.10593(T) = JCM 17270(T)) and strain tADL(T) (= JCM 15066(T) = DSMZ 22187(T)) for which we propose the names Halohasta litorea gen. nov., sp. nov. and Halohasta litchfieldiae sp. nov., respectively.

Halobellus limi sp. nov. and Halobellus salinus sp. nov., isolated from two marine solar salterns.

Two halophilic archaea, strains TBN53(T) and CSW2.24.4(T), were characterized to elucidate their taxonomic status. Strain TBN53(T) was isolated from the Taibei marine solar saltern near Lianyungang city, Jiangsu province, China, whereas strain CSW2.24.4(T) was isolated from a saltern crystallizer in Victoria, Australia. Cells of the two strains were pleomorphic, stained Gram-negative and produced red-pigmented colonies. Strain TBN53(T) was able to grow at 25-55 °C (optimum 45 °C), with 1.4-5.1 M NaCl (optimum 2.6-3.9 M NaCl), with 0-1.0 M MgCl(2) (optimum 0-0.1 M MgCl(2)) and at pH 5.5-9.5 (optimum pH 7.0), whereas strain CSW2.24.4(T) was able to grow at 25-45 °C (optimum 37 °C), with 2.6-5.1 M NaCl (optimum 3.4 M NaCl), with 0.01-0.7 M MgCl(2) (optimum 0.05 M MgCl(2)) and at pH 5.5-9.5 (optimum pH 7.0-7.5). Cells of the two isolates lysed in distilled water. The minimum NaCl concentrations that prevented cell lysis were 8 % (w/v) for strain TBN53(T) and 12 % (w/v) for strain CSW2.24.4(T). The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate, with two glycolipids chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. Trace amounts of other unidentified lipids were also detected. On the basis of 16S rRNA gene sequence analysis, strains TBN53(T) and CSW2.24.4(T) showed 94.1 % similarity to each other and were closely related to Halobellus clavatus TNN18(T) (95.0 and 94.7 % similarity, respectively). Levels of rpoB' gene sequence similarity between strains TBN53(T) and CSW2.24.4(T), and between these strains and Halobellus clavatus TNN18(T) were 88.5, 88.5 and 88.1 %, respectively. The DNA G+C contents of strains TBN53(T) and CSW2.24.4(T) were 69.2 and 67.0 mol%, respectively. The level of DNA-DNA relatedness between strain TBN53(T) and strain CSW2.24.4(T) was 25 %, and these two strains showed low levels of DNA-DNA relatedness with Halobellus clavatus TNN18(T) (30 and 29 % relatedness, respectively). Based on these phenotypic, chemotaxonomic and phylogenetic properties, two novel species of the genus Halobellus are proposed to accommodate these two strains, Halobellus limi sp. nov. (type strain TBN53(T) = CGMCC 1.10331(T) = JCM 16811(T)) and Halobellus salinus sp. nov. (type strain CSW2.24.4(T) = DSM 18730(T) = CGMCC 1.10710(T) = JCM 14359(T)).

Haloquadratum walsbyi: limited diversity in a global pond.

BACKGROUND: Haloquadratum walsbyi commonly dominates the microbial flora of hypersaline waters. Its cells are extremely fragile squares requiring >14%(w/v) salt for growth, properties that should limit its dispersal and promote geographical isolation and divergence. To assess this, the genome sequences of two isolates recovered from sites at near maximum distance on Earth, were compared. PRINCIPAL FINDINGS: Both chromosomes are 3.1 MB in size, and 84% of each sequence was highly similar to the other (98.6% identity), comprising the core sequence. ORFs of this shared sequence were completely synteneic (conserved in genomic orientation and order), without inversion or rearrangement. Strain-specific insertions/deletions could be precisely mapped, often allowing the genetic events to be inferred. Many inferred deletions were associated with short direct repeats (4-20 bp). Deletion-coupled insertions are frequent, producing different sequences at identical positions. In cases where the inserted and deleted sequences are homologous, this leads to variant genes in a common synteneic background (as already described by others). Cas/CRISPR systems are present in C23(T) but have been lost in HBSQ001 except for a few spacer remnants. Numerous types of mobile genetic elements occur in both strains, most of which appear to be active, and with some specifically targetting others. Strain C23(T) carries two ∼6 kb plasmids that show similarity to halovirus His1 and to sequences nearby halovirus/plasmid gene clusters commonly found in haloarchaea. CONCLUSIONS: Deletion-coupled insertions show that Hqr. walsbyi evolves by uptake and precise integration of foreign DNA, probably originating from close relatives. Change is also driven by mobile genetic elements but these do not by themselves explain the atypically low gene coding density found in this species. The remarkable genome conservation despite the presence of active systems for genome rearrangement implies both an efficient global dispersal system, and a high selective fitness for this species.

Halonotius pteroides gen. nov., sp. nov., an extremely halophilic archaeon recovered from a saltern crystallizer.

Strains 1.15.5(T), 2.27.5, 5.24.4 and 6.14.5 were isolated from a solar saltern. They have flattened, rod-shaped cells and are aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media around neutral pH and containing 20-24 % (w/v) (strains 1.15.5(T) and 2.27.5) or 22-24 % (w/v) (5.24.4 and 6.14.5) salts. Mg(2+) was not required. The DNA G+C contents of these isolates were all close to 58 mol%, and DNA-DNA cross-hybridization showed a mean relatedness of 77 %. Their 16S rRNA gene sequences differed by no more than 1.6 % from each other. Phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halorubrum saccharovorum (86.6-87.6 % 16S rRNA gene sequence similarity to the type strain). The only major polar lipid of all four isolates was the sulfated diglycosyl diether lipid S-DGD-1. By phase-contrast microscopy, the long, flattened cells of these strains often displayed a 'wing-like' shape. The phenotypic and phylogenetic data support the placement of these isolates into a novel species in a new genus within the Halobacteriaceae, for which we propose the name Halonotius pteroides gen. nov., sp. nov. The type strain of Halonotius pteroides is 1.15.5(T) (=JCM 14355(T) =CECT 7525(T) =DSM 18729(T)), with the additional reference strains 2.27.5 (=JCM 14356 =DSM 18671), 5.24.4 (=JCM 14357 =DSM 18673) and 6.14.5 (=JCM 14358 =DSM 18692).

Natronomonas moolapensis sp. nov., non-alkaliphilic isolates recovered from a solar saltern crystallizer pond, and emended description of the genus Natronomonas.

Two isolates of non-alkaliphilic, extremely halophilic archaea, with very similar characteristics, were recovered from a marine solar saltern crystallizer. Cells were pleomorphic, motile and Gram-stain-negative and grew on a limited range of carbon sources, with pyruvate being the best substrate. Optimum growth occurred at 18-20 % (w/v) NaCl, pH 6.0-8.5 and 37-40 degrees C. Both isolates possessed typical archaeal lipids, and their 16S rRNA gene sequences were 99.8 % identical. Phylogenetic tree reconstructions indicated that they were most closely related to the haloalkaliphile Natronomonas pharaonis (97.5 % similarity to the type strain), but the different phenotypic properties and low DNA-DNA hybridization values between Nmn. pharaonis DSM 2160(T) and the two isolates suggested that they represent a novel species within the genus Natronomonas. The name Natronomonas moolapensis sp. nov. is proposed for these isolates, with the type strain being 8.8.11(T) (=JCM 14361(T) =CECT 7526(T) =DSM 18674(T)). An emended description of the genus Natronomonas is also provided.

Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeon of Walsby, isolated from saltern crystallizers in Australia and Spain.

Strains C23T and HBSQ001 were isolated from solar salterns and are novel square-shaped, aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media containing 18 % salts at around neutral pH. Mg2+ is not required. The DNA G+C content of both isolates was 46.9 mol% and DNA-DNA cross-hybridization showed a relatedness of 80 %. Their 16S rRNA gene sequences showed only 2 nucleotide differences (99.9 % identity) and phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halogeometricum borinquense PR 3T (91.2 % sequence identity). The major polar glycolipid of both isolates was the sulfated diglycosyl diether lipid S-DGD-1. Electron cryomicrosopy of whole cells revealed similar internal structures, such as gas vesicles and polyhydroxyalkanoate granules, but the cell wall of isolate HBSQ001 displayed a more complex S-layer compared with that of isolate C23T. The phenotypic characterization and phylogenetic data support the placement of isolates C23T and HBSQ001 in a novel species in a new genus within the Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov., sp. nov. The type strain of Haloquadratum walsbyi is C23T (=JCM 12705T=DSM 16854T).

SH1: A novel, spherical halovirus isolated from an Australian hypersaline lake.

A novel halovirus, SH1, with a spherical morphology is described. Isolated from a hypersaline lake, SH1 is divalent, producing clear plaques on Haloarcula hispanica and a natural Halorubrum isolate. Single-step growth curves gave a latent period of 5-6 h and a burst size of around 200 PFU/cell. The host can differentiate to form tight clusters of thick cell-walled forms, and these were shown to be resistant to infection. Purified virions had no visible tail, were about 70 nm in diameter, and displayed a fragile outer capsid layer, possibly with an underlying membrane component. The structural proteins of the virion were analyzed by SDS-PAGE and several were found to be cross-linked, forming protein complexes. The genome was linear, dsDNA, of approximately 30 kb in length. This morphology and linear genome are features not observed in any other euryarchaeal viruses, but have properties similar to the bacterial virus PRD1.

Cultivation of Walsby's square haloarchaeon.

The square haloarchaea of Walsby (SHOW group) dominate hypersaline microbial communities but have not been cultured since their discovery 25 years ago. We show that natural water dilution cultures can be used to isolate members of this group and, once in pure culture, they can be grown in standard halobacterial media. Cells display a square morphology and contain gas vesicles and poly-beta-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was >99% identical to other SHOW group sequences. They prefer high salinities (23-30%), and can grow with a doubling time of 1-2 days in rich media. The ability to culture SHOW group organisms makes it possible to study, in a comprehensive way, the microbial ecology of salt lakes.