Open Issues for Protein Function Assignment in Haloferax volcanii and Other Halophilic Archaea.

BACKGROUND: Annotation ambiguities and annotation errors are a general challenge in genomics. While a reliable protein function assignment can be obtained by experimental characterization, this is expensive and time-consuming, and the number of such Gold Standard Proteins (GSP) with experimental support remains very low compared to proteins annotated by sequence homology, usually through automated pipelines. Even a GSP may give a misleading assignment when used as a reference: the homolog may be close enough to support isofunctionality, but the substrate of the GSP is absent from the species being annotated. In such cases, the enzymes cannot be isofunctional. Here, we examined a variety of such issues in halophilic archaea (class Halobacteria), with a strong focus on the model haloarchaeon Haloferax volcanii. RESULTS: Annotated proteins of Hfx. volcanii were identified for which public databases tend to assign a function that is probably incorrect. In some cases, an alternative, probably correct, function can be predicted or inferred from the available evidence, but this has not been adopted by public databases because experimental validation is lacking. In other cases, a probably invalid specific function is predicted by homology, and while there is evidence that this assigned function is unlikely, the true function remains elusive. We listed 50 of those cases, each with detailed background information, so that a conclusion about the most likely biological function can be drawn. For reasons of brevity and comprehension, only the key aspects are listed in the main text, with detailed information being provided in a corresponding section of the Supplementary Materials. CONCLUSIONS: Compiling, describing and summarizing these open annotation issues and functional predictions will benefit the scientific community in the general effort to improve the evaluation of protein function assignments and more thoroughly detail them. By highlighting the gaps and likely annotation errors currently in the databases, we hope this study will provide a framework for experimentalists to systematically confirm (or disprove) our function predictions or to uncover yet more unexpected functions.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Halomicroarcula amylolytica sp. nov., a novel halophilic archaeon isolated from a salt mine.

A halophilic archaeon named strain LR21T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21T grew at 20-50 °C (optimum, 42 °C), with 8-30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5-9.5 (optimum, pH 7.5-8.5). Mg2+ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T with sequence similarities of 95.3-99.0, 93.0-96.2 and 93.2-95.9 %, respectively, and much lower values to other members. The rpoB' gene sequence similarities between strain LR21T and Hma. limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21T and Hma. limicola YGHS32T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21T and Hma. limicola YGHS32T determined by in silico DNA-DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21T and other members in the genus Halomicroarcula were far below 95 % and the DNA-DNA relatedness values between strain LR21T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21T was 62.0 mol% (genome). The results suggested that strain LR21T represents a novel species of the genus Halomicroarcula, for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21T (=CGMCC 1.13611T=NBRC 113588T).

Reclassification of the genus Natronolimnobius: proposal of two new genera, Natronolimnohabitans gen. nov. to accommodate Natronolimnobius innermongolicus and Natrarchaeobaculum gen. nov. to accommodate Natronolimnobius aegyptiacus and Natronolimnobius sulfurireducens.

The genus Natronolimnobius, currently including four species, is a member of the order Natrialbales, class Halobacteria, and consists of obligately alkaliphilic and extremely halophilic members found exclusively in highly alkaline hypersaline soda lakes. The species were classified into this genus mostly based on phylogenetic analysis of the 16S rRNA gene. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein markers clearly indicates a polyphyletic origin of the species included into this genus, thus warranting its reclassification into three separate genera. We therefore propose to transfer Nlb. innermongolicus (type strain N-1311) to a new genus Natronolimnohabitans as Nlh. innermongolicus comb. nov. and to transfer Nlb. aegyptiacus (type strain JW/NM-HA 15) and Nlb. sulfurireducens (type strain AArc1) to a new genus Natrarchaeobaculum as Nbl. aegyptiacum comb. nov. and Nbl. sulfurireducens comb. nov. The phylogenomic differentiation of these four species is also supported by the ANI/AAI distances and unique phenotypes. The most important physiological differences includes a previously unreported ability for cellulose and xylan utilization in Nlb. baerhuensis, thermophily in Nbl. aegyptiacus and anaerobic sulfur respiration in Nbl. sulfurireducens. We further present an emended description of Natronolimnobius baerhuensis.

Salinibaculum litoreum gen. nov., sp. nov., isolated from salted brown alga Laminaria.

A novel Gram-stain-negative, aerobic and rod-shaped halophilic archaeon, designated HD8-45T, was isolated from the red brine of salted brown alga Laminaria produced at Dalian, PR China. According to the results of 16S rRNA gene and rpoB' gene sequence comparisons, strain HD8-45T showed the highest sequence similarity to the corresponding genes of Salinirussus salinus YGH44T (95.1 and 85.2 % similarities, respectively), Halovenus aranensis EB27T (91.2 and 86.0 % similarities, respectively). The low sequence similarity and the phylogeny implied the novel generic status of strain HD8-45T. Genomic relatedness analyses showed that strain HD8-45T were clearly distinguished from other species in the order Halobacteriales, with average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization values not more than 75.1, 65.6 and 21.5 %. The polar lipid pattern contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids and two minor glycolipids. The two major glycolipids and a minor glycolipid were chromatographically identical to disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. The major respiratory quinones were menaquinone MK-8 and MK-8(H2). The DNA G+C content was 62.0 mol% (Tm) and 61.9 mol% (genome). All these results showed that strain HD8-45T represents a novel species of a new genus in the order Halobacteriales, for which the name Salinibaculum litoreum gen. nov., sp. nov. is proposed. The type strain of Salinibaculum litoreum is HD8-45T (=CGMCC 1.15328T=JCM 31107T).

Natronolimnobius sulfurireducens sp. nov. and Halalkaliarchaeum desulfuricum gen. nov., sp. nov., the first sulfur-respiring alkaliphilic haloarchaea from hypersaline alkaline lakes.

Eight pure cultures of alkaliphilic haloaloarchaea capable of growth by dissimilatory sulfur reduction (previously only shown for neutrophilic haloarchaea) were isolated from hypersaline alkaline lakes in different geographic locations. These anaerobic enrichments, inoculated with sediments and brines, used formate, butyrate and peptone as electron donors and elemental sulfur as an electron acceptor 4 M total Na+ and at pH 9-10. According to 16S rRNA gene sequencing, the isolates fell into two distinct groups. A major group, comprising seven obligate alkaliphilic isolates from highly alkaline soda lakes, represents a new species-level branch within the genus Natronolimnobius (order Natrialbales), while a single moderately alkaliphilic isolate from the less alkaline Searles Lake forms a novel genus-level lineage within the order Haloferacales. The cells of the isolates are either flat rods or coccoid. They are facultative anaerobes using formate or H2 (in the presence of acetate or yeast extract as carbon source), C4-C9 fatty acids or peptone (the major group) as electron donors and either sulfur or DMSO (the major group) as electron acceptors. Aerobic growth is only possible with organic acids and peptone-yeast extract. All isolates are extreme halophiles, growing optimally at 4 M total Na+. On the basis of their unique physiological properties and distinct phylogeny, we propose that the seven isolates from the soda lakes are placed into a novel species, Natronolimnobiussulfurireducens sp. nov. (type strain AArc1T=JCM 30663T=UNIQEM U932T), and the Searles Lake isolate, AArc-SlT, into a new genus and species Halalkaliarchaeum desulfuricum (=JCM 30664T=UNIQEM U999T).

Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes.

An extremely halophilic euryarchaeon, strain HArcel1T, was enriched and isolated in pure culture from the surface brines and sediments of hypersaline athalassic lakes in the Kulunda Steppe (Altai region, Russia) using amorphous cellulose as the growth substrate. The colonies of HArcel1T are pale-orange, and form large zones of cellulose hydrolysis around them. The cells are non-motile cocci of variable size with a thin monolayer cell wall. The isolate is an obligate aerobic heterotroph capable of growth with only three substrates: various forms of insoluble cellulose, xylan and cellobiose. Strain HArcel1T is an extremely halophilic neutrophile, growing within the salinity range from 2.5 to 5 M NaCl (optimum at 3.5-4 M). The core archaeal lipids are dominated by C20-C20 and C25-C20 dialkyl glycerol ethers, in approximately 6:1 proportion. The 16S rRNA and rpoB' gene analysis indicated that HArcel1T forms a separate lineage within the family Haloarculaceae, order Halobacteriales, with the genera Halorhabdus and Halopricus as closest relatives. On the basis of the unique phenotypic properties and distinct phylogeny of the 16S rRNA and rpoB' genes, it is suggested that strain HArcel1T is classified into a new genus and species Halococcoides cellulosivorans gen. nov., sp. nov. (JCM 31941T=UNIQEM U975T).

Extracellular hydrolytic enzymes produced by halophilic bacteria and archaea isolated from hypersaline lake.

The screening of bacteria and archaea from Chott El Jerid, a hypersaline lake in the south of Tunisia, led to the isolation of 68 extremely halophilic prokaryotes growing in media with 15-25% of salt. Assessment of 68 partial 16S rRNA analyzed by amplified rDNA restriction analysis (ARDRA) revealed 15 different bacterial and archaeal taxonomic groups. Based on ARDRA results, phenotypic and hydrolytic activity tests, 20 archaeal and 6 bacterial isolates were selected for sequencing. The halophilic isolates were identified as members of the genera: Salicola, Bacillus, Halorubrum, Natrinema and Haloterrigena. Most of these isolates are able to produce hydrolytic enzymes such as amylase, protease, lipase, cellulase, xylanase, pectinase and some of them showed combined activities. Natrinema genus is an excellent candidate for lipase production. These results indicated that the extremely halophilic archaea and bacteria from Chott El Jerid are a potential source of hydrolytic enzymes and may possess commercial value.

Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic member of the family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes.

Six strains of extremely halophilic and alkaliphilic euryarchaea were enriched and isolated in pure culture from surface brines and sediments of hypersaline alkaline lakes in various geographical locations with various forms of insoluble cellulose as growth substrate. The cells are mostly flat motile rods with a thin monolayer cell wall while growing on cellobiose. In contrast, the cells growing with cellulose are mostly nonmotile cocci covered with a thick external EPS layer. The isolates, designated AArcel, are obligate aerobic heterotrophs with a narrow substrate spectrum. All strains can use insoluble celluloses, cellobiose, a few soluble glucans and xylan as their carbon and energy source. They are extreme halophiles, growing within the range from 2.5 to 4.8M total Na+ (optimum at 4M) and obligate alkaliphiles, with the pH range for growth from 7.5 to 9.9 (optimum at 8.5-9). The core archaeal lipids of strain AArcel5T were dominated by C20-C20 dialkyl glycerol ether (DGE) (i.e. archaeol) and C20-C25 DGE in nearly equal proportion. The 16S rRNA gene analysis indicated that all six isolates belong to a single genomic species mostly related to the genera Saliphagus-Natribaculum-Halovarius. Taking together a substantial phenotypic difference of the new isolates from the closest relatives and the phylogenetic distance, it is concluded that the AArcel group represents a novel genus-level branch within the family Natrialbaceae for which the name Natronobiforma cellulositropha gen. nov., sp. nov. is proposed with AArcel5T as the type strain (JCM 31939T=UNIQEM U972T).

Salinifilum gen. nov., with description of Salinifilum proteinilyticum sp. nov., an extremely halophilic actinomycete isolated from Meighan wetland, Iran, and reclassification of Saccharopolyspora aidingensis as Salinifilum aidingensis comb. nov. and Saccharopolyspora ghardaiensis as Salinifilum ghardaiensis comb. nov.

A Gram-positive, halophilic actinobacterial strain Miq-12T was isolated from Meighan wetland in Iran. Strain Miq-12T was strictly aerobic, catalase positive and oxidase negative. The isolate grew at 12-25 % NaCl, at 30-50 °C and pH 5.5-10.5. The optimum NaCl, temperature and pH for growth were 15-20 %, 40 °C and 7.0-8.0, respectively. The cell wall of strain Miq-12T contained meso-diaminopimelic acid as diagnostic diamino acid and arabinose as whole-cell sugar. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. It synthesized cellular fatty acids of anteiso and iso-branched types, anteiso-C17 : 0, iso-C17:0, iso-C15:0, iso-C16 : 0. The major respiratory quinone was MK-9(H4). The G+C content of its genomic DNA was 72.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Miq-12T belongs to the family Pseudonocardiaceae, constituted a separate clade, and showed the closest phylogenetic similarity to Saccharopolyspora aidingensis TRM 46074T (96.99 %) and Saccharopolyspora ghardaiensis CCUG 63370T (96.92 %). On the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics, a novel genus and species of the family Pseudonocardiaceae, Salinifilum proteinilyticum gen. nov., sp. nov., are proposed. The type strain is Miq-12T (=IBRCM 11033T=LMG 28390T). We also propose that S. aidingensis and S. ghardaiensis should be transferred to this new genus and be named Salinifilum aidingensis comb. nov. and Salinifilum ghardaiensis comb. nov., respectively. The type strain of Salinifilum aidingensis comb. nov. is TRM 46074T (=CCTCCAA 2012014T=JCM 30185T) and the type strain of Salinifilum ghardaiensis comb. nov. is CCUG 63370T (=DSM 45606T=CECT 8304T).

Systematics of haloarchaea and biotechnological potential of their hydrolytic enzymes.

Halophilic archaea, also referred to as haloarchaea, dominate hypersaline environments. To survive under such extreme conditions, haloarchaea and their enzymes have evolved to function optimally in environments with high salt concentrations and, sometimes, with extreme pH and temperatures. These features make haloarchaea attractive sources of a wide variety of biotechnological products, such as hydrolytic enzymes, with numerous potential applications in biotechnology. The unique trait of haloarchaeal enzymes, haloenzymes, to sustain activity under hypersaline conditions has extended the range of already-available biocatalysts and industrial processes in which high salt concentrations inhibit the activity of regular enzymes. In addition to their halostable properties, haloenzymes can also withstand other conditions such as extreme pH and temperature. In spite of these benefits, the industrial potential of these natural catalysts remains largely unexplored, with only a few characterized extracellular hydrolases. Because of the applied impact of haloarchaea and their specific ability to live in the presence of high salt concentrations, studies on their systematics have intensified in recent years, identifying many new genera and species. This review summarizes the current status of the haloarchaeal genera and species, and discusses the properties of haloenzymes and their potential industrial applications.

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats.

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.

Seasonal dynamics of extremely halophilic microbial communities in three Argentinian salterns.

Seasonal sampling was carried out at three Argentinian salterns, Salitral Negro (SN), Colorada Grande (CG) and Guatraché (G), to analyze abiotic parameters and microbial diversity and dynamics. Microbial assemblages were correlated to environmental factors by statistical analyses. Principal component analysis of the environmental data grouped SN and CG samples separately from G samples owing to G's higher pH values and sulfate concentration. Differences in microbial assemblages were also found. Many archaeal sequences belonged to uncultured members of Haloquadratum and Haloquadratum-related genera, with different environmental optima. Notably, nearly half of the archaeal sequences were affiliated to the recently described 'Candidatus Haloredividus' (phylum Nanohaloarchaeota), not previously detected in salt-saturated environments. Most bacterial sequences belonged to Salinibacter representatives, while sequences affiliated to the recently described genus Spiribacter were also found. Seasonal analysis showed at least 40% of the microbiota from the three salterns was prevalent through the year, indicating they are well adapted to environmental fluctuations. On the other hand, a minority of archaeal and bacterial sequences were found to be seasonally distributed. Five viral morphotypes and also eukaryal predators were detected, suggesting different mechanisms for controlling prokaryotic numbers. Notably, Guatraché was the saltern that harbored the highest virus-to-cell ratios reported to date for hypersaline environments.

Analysis of the bacteriorhodopsin-producing haloarchaea reveals a core community that is stable over time in the salt crystallizers of Eilat, Israel.

Stability of microbial communities can impact the ability of dispersed cells to colonize a new habitat. Saturated brines and their halophile communities are presumed to be steady state systems due to limited environmental perturbations. In this study, the bacteriorhodopsin-containing fraction of the haloarchaeal community from Eilat salt crystallizer ponds was sampled five times over 3 years. Analyses revealed the existence of a constant core as several OTUs were found repeatedly over the length of the study: OTUs comprising 52 % of the total cloned and sequenced PCR amplicons were found in every sample, and OTUs comprising 89 % of the total sequences were found in more than one, and often more than two samples. LIBSHUFF and UNIFRAC analyses showed statistical similarity between samples and Spearman's coefficient denoted significant correlations between OTU pairs, indicating non-random patterns in abundance and co-occurrence of detected OTUs. Further, changes in the detected OTUs were statistically linked to deviations in salinity. We interpret these results as indicating the existence of an ever-present core bacteriorhodopsin-containing Eilat crystallizer community that fluctuates in population densities, which are controlled by salinity rather than the extinction of some OTUs and their replacement through immigration and colonization.

Halanaeroarchaeum sulfurireducens gen. nov., sp. nov., the first obligately anaerobic sulfur-respiring haloarchaeon, isolated from a hypersaline lake.

Anaerobic enrichments with acetate as electron donor and carbon source, and elemental sulfur as electron acceptor at 4 M NaCl using anaerobic sediments and brines from several hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in isolation in pure culture of four strains of obligately anaerobic haloarchae growing exclusively by sulfur respiration. Such metabolism has not yet been demonstrated in any known species of Halobacteria, and in the whole archaeal kingdom, acetate oxidation with sulfur as acceptor was not previously demonstrated. The four isolates had nearly identical 16S rRNA gene sequences and formed a novel genus-level branch within the family Halobacteriaceae. The strains had a restricted substrate range limited to acetate and pyruvate as electron donors and elemental sulfur as electron acceptor. In contrast to aerobic haloarchaea, the biomass of anaerobic isolates completely lacked the typical red pigments. Growth with acetate+sulfur was observed between 3-5 M NaCl and at a pH range from 6.7 to 8.0. The membrane core lipids were dominated by archaeols. On the basis of distinct physiological and phylogenetic data, the sulfur-respiring isolates represent a novel species of a new genus in the family Halobacteriaceae, for which the name Halanaeroarchaeaum sulfurireducens gen. nov., sp. nov. is proposed. The type strain of the type species is HSR2T (=JCM 30661T=UNIQEM U935T).

Lentibacillus kimchii sp. nov., an extremely halophilic bacterium isolated from kimchi, a Korean fermented vegetable.

A Gram-positive, aerobic, non-motile and extremely halophilic bacterial strain, designated K9(T), was isolated from kimchi, a Korean fermented food. The strain was observed as endospore-forming rod-shaped cells showing oxidase and catalase activity. It was found to grow at 10.0-30.0 % (w/v) NaCl (optimum, 15.0-20.0 %), pH 7.0-8.0 (optimum, pH 7.5) and 15-40 °C (optimum, 30 °C). The polar lipids of strain K9(T) were identified as phosphatidylglycerol, three unidentified phospholipids and an unidentified glycolipid. The isoprenoid quinone was identified as menaquinone-7. The major cellular fatty acids (>20 % of the total) were found to be anteisio-C15:0 and anteisio-C17:0. The cell wall peptidoglycan composition was determined to contain meso-diaminopimelic acid. The G + C content of genomic DNA was determined to be 48.2 mol %. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolated strain is closely related to Lentibacillus salinarum AHS-1(T) (96.7 % sequence similarity). Based on its phenotypic, chemotaxonomic and phylogenetic data, strain K9(T) is considered to represent a novel species of the genus Lentibacillus, for which the name Lentibacillus kimchii sp. nov., is proposed. The type strain is K9(T) (=KACC 18490(T) = JCM 30234(T)).

A phylogenomic reappraisal of family-level divisions within the class Halobacteria: proposal to divide the order Halobacteriales into the families Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov., and the order Haloferacales into the families, Haloferacaceae and Halorubraceae fam nov.

The evolutionary interrelationships between the archaeal organisms which comprise the class Halobacteria have proven difficult to elucidate using traditional phylogenetic tools. The class currently contains three orders. However, little is known about the family level relationships within these orders. In this work, we have completed a comprehensive comparative analysis of 129 sequenced genomes from members of the class Halobacteria in order to identify shared molecular characteristics, in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can provide reliable evidence, independent of phylogenetic trees, that the species from the groups in which they are found are specifically related to each other due to common ancestry. Here we present 20 CSIs and 31 CSPs which are unique characteristics of infra-order level groups of genera within the class Halobacteria. We also present 40 CSIs and 234 CSPs which are characteristic of Haloarcula, Halococcus, Haloferax, or Halorubrum. Importantly, the CSIs and CSPs identified here provide evidence that the order Haloferacales contains two main groups, one consisting of Haloferax and related genera supported by four CSIs and five CSPs and the other consisting of Halorubrum and related genera supported by four CSPs. We have also identified molecular characteristics that suggest that the polyphyletic order Halobacteriales contains at least two large monophyletic clusters of organisms in addition to the polyphyletic members of the order, one cluster consisting of Haloarcula and related genera supported by ten CSIs and nineteen CSPs and the other group consisting of the members of the genus Halococcus supported by nine CSIs and 23 CSPs. We have also produced a highly robust phylogenetic tree based on the concatenated sequences of 766 proteins which provide additional support for the relationships identified by the CSIs and CSPs. On the basis of the phylogenetic analyses and the identified conserved molecular characteristics presented here, we propose a division of the order Haloferacales into two families, an emended family Haloferacaceae and Halorubraceae fam. nov. and a division of the order Halobacteriales into three families, an emended family Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov.

Patterns and determinants of halophilic archaea (class halobacteria) diversity in tunisian endorheic salt lakes and sebkhet systems.

We examined the diversity and community structure of members of the halophilic Archaea (class Halobacteria) in samples from central and southern Tunisian endorheic salt lakes and sebkhet (also known as sebkha) systems using targeted 16S rRNA gene diversity survey and quantitative PCR (qPCR) approaches. Twenty-three different samples from four distinct locations exhibiting a wide range of salinities (2% to 37%) and physical characteristics (water, salt crust, sediment, and biofilm) were examined. A total of 4,759 operational taxonomic units at the 0.03 (species-level) cutoff (OTU0.03s) belonging to 45 currently recognized genera were identified, with 8 to 43 genera (average, 30) identified per sample. In spite of the large number of genera detected per sample, only a limited number (i.e., 2 to 16) usually constituted the majority (≥80%) of encountered sequences. Halobacteria diversity showed a strong negative correlation to salinity (Pearson correlation coefficient = -0.92), and community structure analysis identified salinity, rather than the location or physical characteristics of the sample, as the most important factor shaping the Halobacteria community structure. The relative abundance of genera capable of biosynthesis of the compatible solute(s) trehalose or 2-sulfotrehalose decreased with increasing salinities (Pearson correlation coefficient = -0.80). Indeed, qPCR analysis demonstrated that the Halobacteria otsB (trehalose-6-phosphatase)/16S rRNA gene ratio decreases with increasing salinities (Pearson correlation coefficient = -0.87). The results highlight patterns and determinants of Halobacteria diversity at a previously unexplored ecosystem and indicate that genera lacking trehalose biosynthetic capabilities are more adapted to growth in and colonization of hypersaline (>25% salt) ecosystems than trehalose producers.

Diversity of extremely halophilic cultivable prokaryotes in Mediterranean, Atlantic and Pacific solar salterns: Evidence that unexplored sites constitute sources of cultivable novelty.

The culturable fraction of aerobic, heterotrophic and extremely halophilic microbiota retrieved from sediment and brine samples of eight sampling sites in the Mediterranean, Canary Islands and Chile was studied by means of a tandem approach combining large-scale cultivation, MALDI-TOF MS targeting whole cell biomass, and phylogenetic reconstruction based on 16S rRNA gene analysis. The approach allowed the identification of more than 4200 strains and a comparison between different sampling sites. The results indicated that the method constituted an excellent tool for the discovery of taxonomic novelty. Four new genera and nine new species could be identified within the archaeal family Halobacteriaceae, as well as one new bacterial species, and a representative of Salinibacter ruber phylotype II, a group that had been refractory to isolation for the last fifteen years. Altogether, the results indicated that in order to provide better yields for the retrieval of novel taxa from the environment, performance of non-redundant environment sampling is recommended together with the screening of large sets of strains.

Phylogenomic analyses and molecular signatures for the class Halobacteria and its two major clades: a proposal for division of the class Halobacteria into an emended order Halobacteriales and two new orders, Haloferacales ord. nov. and Natrialbales ord. nov., containing the novel families Haloferacaceae fam. nov. and Natrialbaceae fam. nov.

The Halobacteria constitute one of the largest groups within the Archaea. The hierarchical relationship among members of this large class, which comprises a single order and a single family, has proven difficult to determine based upon 16S rRNA gene trees and morphological and physiological characteristics. This work reports detailed phylogenetic and comparative genomic studies on >100 halobacterial (haloarchaeal) genomes containing representatives from 30 genera to investigate their evolutionary relationships. In phylogenetic trees reconstructed on the basis of 32 conserved proteins, using both neighbour-joining and maximum-likelihood methods, two major clades (clades A and B) encompassing nearly two-thirds of the sequenced haloarchaeal species were strongly supported. Clades grouping the same species/genera were also supported by the 16S rRNA gene trees and trees for several individual highly conserved proteins (RpoC, EF-Tu, UvrD, GyrA, EF-2/EF-G). In parallel, our comparative analyses of protein sequences from haloarchaeal genomes have identified numerous discrete molecular markers in the form of conserved signature indels (CSI) in protein sequences and conserved signature proteins (CSPs) that are found uniquely in specific groups of haloarchaea. Thirteen CSIs in proteins involved in diverse functions and 68 CSPs that are uniquely present in all or most genome-sequenced haloarchaea provide novel molecular means for distinguishing members of the class Halobacteria from all other prokaryotes. The members of clade A are distinguished from all other haloarchaea by the unique shared presence of two CSIs in the ribose operon protein and small GTP-binding protein and eight CSPs that are found specifically in members of this clade. Likewise, four CSIs in different proteins and five other CSPs are present uniquely in members of clade B and distinguish them from all other haloarchaea. Based upon their specific clustering in phylogenetic trees for different gene/protein sequences and the unique shared presence of large numbers of molecular signatures, members of clades A and B are indicated to be distinct from all other haloarchaea because of their uniquely shared evolutionary histories. Based upon these results, it is proposed that clades A and B be recognized as two new orders, Natrialbales ord. nov. and Haloferacales ord. nov., within the class Halobacteria, containing the novel families Natrialbaceae fam. nov. and Haloferacaceae fam. nov. Other members of the class Halobacteria that are not members of these two orders will remain part of the emended order Halobacteriales in an emended family Halobacteriaceae.