Using the pan-genomic framework for the discovery of genomic islands in the haloarchaeon Halorubrum ezzemoulense.

In this study, we use pan-genomics to characterize the genomic variability of the widely dispersed halophilic archaeal species Halorubrum ezzemoulense (Hez). We include a multi-regional sampling of newly sequenced, high-quality draft genomes. The pan-genome graph of the species reveals 50 genomic islands that represent rare accessory genetic capabilities available to members. Most notably, we observe rearrangements that have led to the insertion/recombination/replacement of mutually exclusive genomic islands in equivalent genome positions ("homeocassettes"). These conflicting islands encode for similar functions, but homologs from islands located between the same core genes exhibit high divergence on the amino acid level, while the neighboring core genes are nearly identical. Both islands of a homeocassette often coexist in the same geographic location, suggesting that either island may be beyond the reach of selective sweeps and that these loci of divergence between Hez members are maintained and persist long term. This implies that subsections of the population have different niche preferences and rare metabolic capabilities. After an evaluation of the gene content in the homeocassettes, we speculate that these islands may play a role in the speciation, niche adaptability, and group selection dynamics in Hez. Though homeocassettes are first described in this study, similar replacements and divergence of genes on genomic islands have been previously reported in other Haloarchaea and distantly related Archaea, suggesting that homeocassettes may be a feature in a wide range of organisms outside of Hez.IMPORTANCEThis study catalogs the rare genes discovered in strains of the species Halorubrum ezzemoulense (Hez), an obligate halophilic archaeon, through the perspective of its pan-genome. These rare genes are often found to be arranged on islands that confer metabolic and transport functions and contain genes that have eluded previous studies. The discovery of divergent, but homologous islands occupying equivalent genome positions ("homeocassettes") in different genomes, reveals significant new information on genome evolution in Hez. Homeocassette pairs encode for similar functions, but their dissimilarity and distribution imply high rates of recombination, different specializations, and niche preferences in Hez. The coexistence of both islands of a homeocassette pair in multiple environments demonstrates that both islands are beyond the reach of selective sweeps and that these genome content differences between strains persist long term. The switch between islands through recombination under different environmental conditions may lead to a greater range of niche adaptability in Hez.

Halorubrum salinarum sp. nov., an extremely halophilic archaeon isolated from a saturated brine pond of a saltern.

A novel extremely halophilic archaeon, strain RHB-CT, was isolated from a saturated brine pond of a solar saltern in Bolinao, Pangasinan, Philippines. Colonies were orange-red-pigmented, smooth, convex and round on a solid modified growth medium containing 25 % (w/v) of total salts. Cells of strain RHB-CT on the solid modified growth medium were ovoid-shaped (0.89-2.66 µm long), while the cells in a liquid modified growth medium were rod-shaped (1.53-5.65 µm long and 0.45-1.03 µm wide). The strain was Gram-stain-negative, motile and strictly aerobic. Strain RHB-CT grew with NaCl concentrations ranging from 10 to 30 % (w/v; optimum, 20-25 %), at pH 6.5-8.5 (optimum, pH 7.0-7.5) and at 20-55 °C (optimum, 40-45 °C). Furthermore, the strain grew even in the absence of Mg2+; however, when supplemented with Mg2+, growth was observed optimally at 0.2-0.4 M Mg2+. The 16S rRNA gene phylogeny inferred that the strain is a member of the genus Halorubrum and was related to Halorubrum xinjiangense CGMCC 1.3527T (99.0 %), Halorubrum sodomense DSM 3755T (98.8 %), Halorubrum coriense Ch2T (98.8 %), Halorubrum trapanicum NRC 34021T (98.4 %) and Halorubrum distributum JCM 9100T (98.1 %). The rpoB' gene sequences also showed that strain RHB-CT is related to Hrr. xinjiangense JCM 12388T (97.1 %), Hrr. distributum JCM 9100T (97.1 %), Hrr. coriense JCM 9275T (96.5 %), Hrr. californiense JCM 14715T (96.5 %), Hrr. trapanicum JCM 10477T (96.3%), Hrr. sodomense JCM 8880T (96.2%) and Hrr. tebenquichense DSM 14210T (95.6 %). The DNA G+C content of strain RHB-CT was 68.7 mol% (genome). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain RHB-CT and the closely related species of Halorubrum were below 40 and 90 %, respectively, which are far below the thresholds to delineate a new species. The polar lipids of strain RHB-CT were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and sulfated mannosyl glycosyl diether. Based on dDDH and ANI values, and the significant morphological and physiological differences from known taxa, it is hereby suggested that strain RHB-CT represents a novel species of the genus Halorubrum, for which the name Halorubrum salinarum sp. nov. is proposed. The type strain is RHB-CT (=KCTC 4274T=CMS 2103T).

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum, Halorubrum, and Halolamina, isolated from solar salt.

Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220, Halorubrum sp. CBA1229, and Halolamina sp. CBA1230, which are facultatively oligotrophic haloarchaea, were isolated from solar salt by culture under oligotrophic culture conditions. The complete genomes of strains CBA1220, CBA1229, and CBA1230 were sequenced and were found to contain 3,175,875, 3,582,278, and 3,465,332 bp, with a G + C content of 68.25, 67.66, and 66.75 mol %, respectively. In total, 60, 36, and 33 carbohydrate-active enzyme genes were determined in the respective strains. The strains harbored various genes encoding stress-tolerance proteins, including universal stress proteins, cold-shock proteins, and rubrerythrin and rubrerythrin-related proteins. The genome data produced in this study will facilitate further research to improve our understanding of other halophilic strains and promote their industrial application.

Comparative diversity analysis of halophiles at two polar saltern systems in Indramayu, West Java, Indonesia.

Successive microbes in solar salt ponds are essential since it is well correlated with the quality of salt produced. This research aimed to analyse the microbial diversity of the solar salt ponds in Indonesia, which use high-density polyethylene in the ponds. There are two systems, that is, an integrated open system (In-system) and a closed system (Tt-system). The In-system uses seawater while the Tt-system uses seawater from the saline artesian well. Results showed that the In-system had richer microbes than the Tt-system. Both systems shared similar halophilic microbes profile. Ponds with low salinity (3-4 Be) had very low archaea, that is, 0·2 and 0·7% for the In-system and Tt-system respectively and were dominantly inhabited by phylum Proteobacteria. In the pond with high salinity, that is, 25 Be, both systems were dominated by the phylum Euryarchaeota, family Halobacteriaceae, and genera Halorubrum was dominantly found in In25 ponds and Tt25 ponds. Even though the two systems use the same parent seawater, that is, the Java Sea and share similar microbial composition at the phylum level, we found the dominance identified microbes in both systems were different.

Interaction of two strongly divergent archaellins stabilizes the structure of the Halorubrum archaellum.

Halophilic archaea from the genus Halorubrum possess two extraordinarily diverged archaellin genes, flaB1 and flaB2. To clarify roles for each archaellin, we compared two natural Halorubrum lacusprofundi strains: One of them contains both archaellin genes, and the other has the flaB2 gene only. Both strains synthesize functional archaella; however, the strain, where both archaellins are present, is more motile. In addition, we expressed these archaellins in a Haloferax volcanii strain from which the endogenous archaellin genes were deleted. Three Hfx. volcanii strains expressing Hrr. lacusprofundi archaellins produced functional filaments consisting of only one (FlaB1 or FlaB2) or both (FlaB1/FlaB2) archaellins. All three strains were motile, although there were profound differences in the efficiency of motility. Both native and recombinant FlaB1/FlaB2 filaments have greater thermal stability and resistance to low salinity stress than single-component filaments. Functional supercoiled Hrr. lacusprofundi archaella can be composed of either single archaellin: FlaB2 or FlaB1; however, the two divergent archaellin subunits provide additional stabilization to the archaellum structure and thus adaptation to a wider range of external conditions. Comparative genomic analysis suggests that the described combination of divergent archaellins is not restricted to Hrr. lacusprofundi, but is occurring also in organisms from other haloarchaeal genera.

Bioprospecting and characterization of pigmented halophilic archaeal strains from Algerian hypersaline environments with analysis of carotenoids produced by Halorubrum sp. BS2.

A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena, and Halopiger. Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 µg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human-pathogenic strains and four fish-pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities.

Genome-based analyses reveal a synonymy among Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996, Halorubrum terrestre Ventosa et al. 2004, Halorubrum arcis Xu et al. 2007 and Halorubrum litoreum Cui et al. 2007. Emended description of Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996.

A comparative taxonomic study of Halorubrum distributum, Halorubrum terrestre, Halorubrum arcis and Halorubrum litoreum was carried out using different approaches, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), phylogenomic analysis based on the comparison of the core genome, orthologous average nucleotide identity (OrthoANI), Genome-to-Genome Distance Calculator (GGDC), synteny plots and polar lipid profile (PLP). The MLSA study, using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and the phylogenomic analysis based on 1347 core translated gene sequences obtained from their genomes showed that Halorubrum distributum JCM 9100T, Halorubrum terrestre JCM 10247T, Halorubrum arcis JCM 13916T and Halorubrum litoreum JCM 13561T formed a robust cluster, clearly separated from the rest of species of the genus Halorubrum. The OrthoANI and digital DDH values, calculated by the GGDC, showed percentages among Hrr. distributum JCM 9100T, Hrr. terrestre JCM 10247T, Hrr. arcis JCM 13916T and Hrr. litoreum JCM 13561T that ranged from 98.1 to 97.5 %, and 84.0 to 78.0 %, respectively, while these values among those strains and the type strains of their most related species of Halorubrum were equal or lower than 90.8 and 41.2 %, respectively. Moreover, degree of synteny across the four genomes was very high, especially between the genomes of Halorubrum litoreum JCM 13561T and Halorubrum arcis JCM 13916T. In addition, the PLP is quite similar among the four strains studied, showing a common pattern typical of the neutrophilic species of the genus Halorubrum. Overall, these data show that Hrr. distributum, Hrr. terrestre, Hrr. arcis and Hrr. litoreum constitute a single species. Thus, the latter three should be considered as later, heterotypic synonyms of Hrr. distributum based on the rules for priority of names. We propose an emended description of Hrr. distributum, including the features of Hrr. terrestre, Hrr. arcis and Hrr. litoreum.

Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine.

A pleomorphic and non-motile halophilic archaeon forming light-red pigmented colonies, strain ZC67T, was isolated from the Yuanyongjing Salt Mine, Yunnan, China. Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain ZC67T belongs to the genus Halorubrum and is closely related to the species of Halorubrum (Hrr.) saccharovorum JCM 8865T, Hrr. persicum C49T, Hrr. halophilum B8T, Hrr. lipolyticum 9-3T, Hrr. salsamenti Y69T and Hrr. depositum Y78T with 16S rRNA gene sequence similarities of 99.0%, 98.7%, 98.5%, 98.4%, 98.1% and 97.7%, respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain ZC67T and its close relatives were less than 90.5% and 89.3%, respectively. In silico DNA-DNA hybridization (DDH) analysis showed that DNA-DNA relatedness between strain ZC67T and its relatives is less than 45%. Values of ANI, AAI and in silico DDH were clearly below the thresholds used for the delineation of a new species. The major polar lipids of strain ZC67T were similar to other neutrophilic members in the genus Halorubrum containing phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl-glucosyl-glycerol diether-1. The DNA G+C content was determined to be 66.3 mol% (based on the draft genome). Combined with other diagnostic characteristics, e.g. phenotypic and chemotaxonomic differences, strain ZC67T is concluded to represent a novel species in the genus Halorubrum, for which the name Halorubrum amylolyticum sp. nov. is proposed. The type strain is ZC67T (=CGMCC 1.15718T = JCM 31850T).

Halorubrum glutamatedens sp. nov., a Halophilic Archaeon Isolated from a Rock Salt.

An extremely halophilic archaeon, strain ZY8T, was isolated from a rock salt of Yunnan salt mine. It was able to grow at 12-30% (w/v) NaCl (optimum, 15-20%), pH 7.0-9.0 (optimum, pH 8.5), and 20-45 °C (optimum, 42 °C). Sequence similarity search of its 16S rRNA gene showed that strain ZY8T belonged to the genus Halorubrum, and it is closely related to species of H. aethiopicum SAH-A6T (98.6%), H. aquaticum EN-2T (98.6%), and H. halodurans Cb34T (98.5%), respectively. Strain ZY8T contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate as its major phospholipids, and a sulfated diglycosyl diether as its major glycolipid. The DNA G+C content was 66.7 mol%. DNA-DNA relatedness between strains ZY8T and closely related species were far below 70%. Based on the phenotypic and phylogenetic analyses, it is proposed that strain ZY8T represents a novel species of the genus Halorubrum, for which the name Halorubrum glutamatedens sp. nov. is proposed. The type strain is ZY8T (=CGMCC 1.16026T=NBRC 112866T).

Halorubrum chaoviator Mancinelli et al. 2009 is a later, heterotypic synonym of Halorubrum ezzemoulense Kharroub et al. 2006. Emended description of Halorubrum ezzemoulense Kharroub et al. 2006.

A polyphasic comparative taxonomic study of Halorubrum ezzemoulense Kharroub et al. 2006, Halorubrum chaoviator Mancinelli et al. 2009 and eight new Halorubrum strains related to these haloarchaeal species was carried out. Multilocus sequence analysis using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and phylogenetic analysis based on the 757 core protein sequences obtained from their genomes showed that Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator Halo-G*T (=DSM 19316T) and the eight Halorubrum strains formed a robust cluster, clearly separated from the remaining species of the genus Halorubrum. The orthoANI value and digital DNA-DNA hybridization value, calculated by the Genome-to-Genome Distance Calculator (GGDC), showed percentages among Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator DSM 19316T and the eight Halorubrum strains ranging from 99.4 to 97.9 %, and from 95.0 to 74.2 %, respectively, while these values for those strains and the type strains of the most closely related species of Halorubrum were 88.7-77.4 % and 36.1-22.3 %, respectively. Although some differences were observed, the phenotypic and polar lipid profiles were quite similar for all the strains studied. Overall, these data show that Hrr. ezzemoulense, Hrr. chaoviator and the eight new Halorubrum isolates constitute a single species. Thus, Hrr. chaoviator should be considered as a later, heterotypic synonym of Hrr. ezzemoulense. We propose an emended description of Hrr. ezzemoulense, including the features of Hrr. chaoviator and those of the eight new isolates.

Genomic variation and biogeography of Antarctic haloarchaea.

BACKGROUND: The genomes of halophilic archaea (haloarchaea) often comprise multiple replicons. Genomic variation in haloarchaea has been linked to viral infection pressure and, in the case of Antarctic communities, can be caused by intergenera gene exchange. To expand understanding of genome variation and biogeography of Antarctic haloarchaea, here we assessed genomic variation between two strains of Halorubrum lacusprofundi that were isolated from Antarctic hypersaline lakes from different regions (Vestfold Hills and Rauer Islands). To assess variation in haloarchaeal populations, including the presence of genomic islands, metagenomes from six hypersaline Antarctic lakes were characterised. RESULTS: The sequence of the largest replicon of each Hrr. lacusprofundi strain (primary replicon) was highly conserved, while each of the strains' two smaller replicons (secondary replicons) were highly variable. Intergenera gene exchange was identified, including the sharing of a type I-B CRISPR system. Evaluation of infectivity of an Antarctic halovirus provided experimental evidence for the differential susceptibility of the strains, bolstering inferences that strain variation is important for modulating interactions with viruses. A relationship was found between genomic structuring and the location of variation within replicons and genomic islands, demonstrating that the way in which haloarchaea accommodate genomic variability relates to replicon structuring. Metagenome read and contig mapping and clustering and scaling analyses demonstrated biogeographical patterning of variation consistent with environment and distance effects. The metagenome data also demonstrated that specific haloarchaeal species dominated the hypersaline systems indicating they are endemic to Antarctica. CONCLUSION: The study describes how genomic variation manifests in Antarctic-lake haloarchaeal communities and provides the basis for future assessments of Antarctic regional and global biogeography of haloarchaea.

Isolation and identification of two extremely halophilic archaea from sebkhas in the Algerian Sahara.

In Algeria, many salt lakes are to be found spread from southern Tunisia up to the Atlas Mountains in northern Algeria. Oum Eraneb and Ain El beida sebkhas (salt lakes), are located in the Algerian Sahara. The aim of this study was to explore the diversity of the halobacteria in this type of habitats. The physicochemical properties of these shallow saline environments were examined and compared with other hypersaline and marine ecosystems. Both sites were relatively alkaline with a pH around 8.57- 8.74 and rich in salt at 13% and 16% (w/v) salinity for Oum Eraneb and Ain El beida, respectively, with dominant ions of sodium and chloride. The microbial approach revealed the presence of two halophilic archaea, strains JCM13561 and A33T in both explored sebkhas. Growth occurred between 10 and 25% (w/v) NaCl and the isolates grow optimally at 20% (w/v) NaCl. The pH range for growth was 6 to 9.5 with an optimum at pH 7.5 for the first strain and 7 to 9.5 with an optimum pH at 8.5-9 for the second strain. On the basis of 16S rRNA gene sequence analysis, strains JCM13561 and A33T were most closely related to Halorubrum litoreum and Natronorubrum bangense (99% and 96% similarity, respectively).

Halorubrum aethiopicum sp. nov., an extremely halophilic archaeon isolated from commercial rock salt.

A novel extremely halophilic archaeon, designated SAH-A6T, was isolated from a sample of commercial rock salt in Ethiopia. Cells of SAH-A6T were aerobic and pleomorphic. The strain was able to grow at concentrations of 15-30 % (w/v) NaCl (optimum 20-25 % NaCl), at pH 6.0-9.0 (optimum pH 7.0) and in a temperature range of 30-55 °C (optimum 37-45 °C). Mg2+ was not required for growth of SAH-A6T cells. On the basis of 16S rRNA gene sequence analysis, strain SAH-A6T was closely related to Halorubrum halodurans Cb34T (99.1 %), Halorubrum rubrum YC87T (98.9 %), Halorubrum aquaticum EN-2T (98.7 %), Halorubrum cibi JCM 15757T (98.4 %), Halorubrum luteum CGSA15T (97.3 %), Halorubrum lipolyticum 9-3T (97.1 %), Halorubrum tibetense 8W8T (97.1 %), Halorubrum kocurii JCM 1478T (97.1 %), Halorubrum halophilum B8T (97.0 %) and Halorubrum persicum C49T (97.0 %). Phylogenetic analysis based on the rpoB' gene sequences showed that strain SAH-A6T was closely related to Hrr. halodurans Cb34T (99.7 %), Hrr. aquaticum JCM 14031T (99.3 %) and other members of the genus Halorubrum (<99.0 %). The DNA G+C content of the strain was 68.0 mol%. DNA-DNA hybridization between strain SAH-A6T and the most closely related members of the genus Halorubrum were below 55 %, suggesting that the new isolate constitutes a different genospecies. On the bases of chemotaxonomic, phenotypic and genotypic data, strain SAH-A6T (=KCCM 43215T=JCM 31519T) represents a novel species of the genus Halorubrum, for which the name Halorubrumaethiopicum sp. nov. is proposed.

Halorubrum salsamenti sp. nov., a Novel Halophilic Archaeon Isolated from a Brine of Salt Mine.

A non-motile, spherical or oval extremely halophilic archaeon, strain Y69T, was isolated from a brine of the Yunnan salt mine, China. Colonies on JCM 168 agar plate were round (1-2 mm in diameter), moist, and orange-pigmented. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence showed that the isolate belonged to the species of the genus Halorubrum, with a close relationship to Halorubrum aidingense 31-hongT (98.5%), Halorubrum lacusprofundi ATCC 49239T (98.2%), and Halorubrum kocurii BG-1T (98.0%). The major polar lipids of strain Y69T were phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl diether. Strain Y69T grew in 15-30% (w/v) NaCl. The temperature and pH ranges for growth were 25-50 °C and 6.5-9.0, respectively. Optimal growth occurred at 20% (w/v) NaCl, 42 °C, and pH 8.0. Mg2+ was required for growth. The DNA G+C content was determined to be 65.1 mol% by the thermal denaturation method. DNA-DNA hybridization values between strain Y69T and the closely related species were lower than 70%. Based on the data presented in this study, strain Y69T represents a novel species for which the name Halorubrum salsamenti sp. nov. is proposed. The type of the strain is Y69T (=CGMCC 1.15455T = JCM 31270T).

Halorubrum trueperi sp. nov., a halophilic archaeon isolated from a salt mine.

A novel, extremely halophilic archaeon, strain Y73T, was isolated from a salt mine in Yunnan, China. Colonies formed on solid medium were circular (2-3 mm in diameter), smooth, orange, glistening and convex (~1 mm in elevation). Cells were Gram-stain-negative, non-motile and pleomorphic. Mg2+ was required for growth (optimum at 0.05 M). Optimal growth was observed at 20 % (w/v) NaCl, 42-45 °C and pH 7.5-8.5 under aerobic conditions. 16S rRNA gene sequence comparison showed that strain Y73T was closely related to Halorubrum halophilum B8T (similarity: 98.1 %), Halorubrum lipolyticum 9-3T (97.9 %) and Halorubrum saccharovorum JCM 8865T (97.6 %). Levels of rpoB' gene sequence similarity between strain Y73T and H. halophilum B8T, H. lipolyticum 9-3T and H. saccharovorum JCM 8865T were 93.6, 93.8 and 94.7 %, respectively. DNA-DNA relatedness between strain Y73T and H. halophilum B8T was 38.7±0.5 %, while that between strain Y73T and H. saccharovorum JCM 8865T was 31.0±0.9 % . The DNA G+C content of strain Y73T was 61.9 mol%. The major polar lipids of strain Y73T were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate as phospholipids, and sulfated diglycosyl diether-1 as glycolipid. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain Y73T represents a novel species, for which the name Halorubrum trueperi sp. nov is proposed. The type strain is Y73T (=CGMCC 1.15503T=JCM 31271T).

Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.

No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.

Halorubrum pallidum sp. nov., an extremely halophilic archaeon isolated from a subterranean rock salt.

An extremely halophilic archaeon, strain PJ61T, was isolated from a subterranean rock salt of Yuanyongjing Salt Mine, Yunnan, China. Colonies were pale, smooth, convex, and round (1.0-2.0 mm in diameter) on nutrient agar plates. Cells of strain PJ61T were spherical or oval , stained Gram-negative, and were non-motile. Optimal growth was observed with 3.4 M NaCl and at 38 °C in aerobic conditions. Mg2+ was required for growth. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain PJ61T belonged to the genus Halorubrum and was closely related to Halorubrum laminariae R60T (98.3 % 16S rRNA gene sequence similarity), Halorubrum salinum GX71T (98.2 %) and other species of the genus Halorubrum (<98 %). Sequence similarities of rpoB' gene and ef-2 gene between strain PJ61T and the species of the genus Halorubrum also showed that strain PJ61T was closely related to strain Halorubrum salinum GX71T (93.4 % for rpoB'and 94.8 % for ef-2). The DNA-DNA relatedness between strains PJ61T and Halorubrum laminariae R60T was 33±0.5 %, while it was 37±0.4 % for Halorubrum salinum GX71T. The DNA G+C content of strain PJ61T was 65.1 mol%. The major polar lipids of strain PJ61T consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain PJ61T represents a novel species of the genus Halorubrum, for which the name Halorubrum pallidum sp. nov. is proposed. The type strain is PJ61T (=CGMCC 1.15212T =JCM 30955T).

Halorubrum halodurans sp. nov., an extremely halophilic archaeon isolated from a hypersaline lake.

Two extremely halophilic archaea, strains Cb34T and C170, belonging to the genus Halorubrum, were isolated from the brine of the hypersaline lake Aran-Bidgol in Iran. Cells of the two strains were motile, pleomorphic rods, stained Gram-variable and produced red-pigmented colonies. Strains Cb34T and C170 required 25 % (w/v) salts, pH 7.0 and 37 °C for optimal growth under aerobic conditions; 0.3 M Mg2+ was required. Cells of both isolates were lysed in distilled water and hypotonic treatment with < 10 % NaCl provoked cell lysis. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that these two strains were closely related to Halorubrum cibi B31T (98.8 %) and other members of the genus Halorubrum. In addition, studies based on the rpoB' gene revealed that strains Cb34T and C170 are placed among the species of Halorubrum and are closely related to Halorubrum cibi B31T, with rpoB' gene sequence similarity less than or equal to 95.7 %. The polar lipid patterns of both strains consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The DNA G+C content was 62.1-62.4 mol%. DNA-DNA hybridization studies confirmed that strains Cb34T and C170 constitute a distinct species. Data obtained in this study show that the two strains represent a novel species, for which the name Halorubrum halodurans sp. nov. is proposed. The type strain is Cb34T ( = CECT 8745T = IBRC-M 10233T).

Halorubrum rutilum sp. nov. isolated from a marine solar saltern.

A halophilic archaeal strain, YJ-18-S1(T), was isolated from Yangjiang marine solar saltern, Guangxi Province, China. Cells were pleomorphic, stained Gram-negative and formed red-pigmented colonies on agar plates. Strain YJ-18-S1(T) was able to grow at 20-55 °C (optimum 37 °C), at 0.9-4.8 M NaCl (optimum 2.6 M NaCl), at 0.005-1.0 M MgCl2 (optimum 0.3 MgCl2) and at pH 5.5-8.5 (optimum pH 7.0). The cells were lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The 16S rRNA gene and rpoB' gene of strain YJ-18-S1(T) were phylogenetically related to the corresponding genes of Halorubrum members (94.3-98.0 and 86.7-96.1 % similarities, respectively). The DNA G+C content of strain YJ-18-S1(T) was 66.2 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YJ-18-S1(T) (=CGMCC 1.12554(T) = JCM 30030(T)) represents a new species of Halorubrum, for which the name Halorubrum rutilum sp. nov. is proposed.

Halorubrum yunnanense sp. nov., isolated from a subterranean salt mine.

Two halophilic archaeal strains, Q85(T) and Q86, were isolated from a subterranean salt mine in Yunnan, China. Cells were rod-shaped, Gram-stain-negative and motile. Colonies were red, smooth, convex and round (1.0-2.0 mm in diameter). The orthologous 16S rRNA and rpoB9 gene sequences of these two strains were almost identical (99.5 and 99.7 % similarities). Their closest relatives were Halorubrum kocurii BG-1(T) (98.0-98.1 % 16S rRNA gene sequence similarity), Halorubrum aidingense 31-hong(T) (97.6­97.7 %) and Halorubrum lipolyticum 9-3T (97.5-97.6 %). The level of DNA-DNA relatedness between strains Q85(T) and Q86 was 90 %, while that between Q85(T) and other related Halorubrum strains was less than 30 % (29 % for H. kocurii BG-1(T), 25 % for H. aidingense 31-hong(T) and 22 % for H. lipolyticum 9-3(T)). Optimal growth of the two novel strains was observed with 20 % (w/v) NaCl and at 42­45 8C under aerobic conditions, with a slight difference in optimum Mg2+ concentration (0.7 M for Q85(T),0.5 M for Q86) and a notable difference in optimum pH (pH 7.5 for Q85(T), pH 6.6 for Q86). Anaerobic growth occurred with nitrate, but not with L-arginine or DMSO. The major polar lipids of the two strains were identical, including phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated diglycosyl diether, which are the major lipids of the genus Halorubrum. The G+C contents of strains Q85(T) and Q86 were 66.3 and 66.8 %, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic properties of strains Q85(T) and Q86, a novel species, Halorubrum yunnanense sp. nov., is proposed. The type strain is Q85(T) (=5CGMCC 1.15057(T)=JCM 30665(T)).