Halococcus salsus sp. nov., a novel halophilic archaeon isolated from rock salt.

Two pink-pigmented halophilic archaea, designated strains ZJ1T and J81, were isolated from rock salt of Yunnan Salt Mine, China, and commercial salt imported from Bolivia, respectively. Cells were non-motile, coccoid, approximately 0.8-1.6 µm in diameter, stained Gram-negative and often occurred in pairs. Colonies were wet, opaque and smooth-edged. Strain ZJ1T grew optimally with 20 % (w/v) NaCl, at pH 7.5 and at 38-40 °C, which was the same as for strain J81. 16S rRNA gene sequence similarity between strains ZJ1T and J81 was 99.7 %. Sequence similarity searches based on the 16S rRNA gene and cell morphology suggested that strains ZJ1T and J81 belong to the genus Halococcus in the family Halococcaceae. The major polar lipids of the type strain, ZJ1T, were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated diglycosyl-diether-1. The profile of polar lipids, cell shape, motility and lack of lysis of cells in distilled water show that strains ZJ1T and J81 were similar to other members of the genus Halococcus. Strain ZJ1T shared the highest 16S rRNA gene and rpoB' gene sequence similarities of 99.0 and 95.3 % with Halococcus hamelinensis 100A6T, respectively, followed by less than 94.6 % with sequences of other species in the genus Halococcus. DNA-DNA relatedness between strains ZJ1T and J81 was 90.1±0.7 %, while 27±0.5 % was found between strain ZJ1T and H. hamelinensis JCM 12892T (=100A6T), and 29.0±0.5 % between strains J81 and H. hamelinensis JCM 12892T. The DNA G+C content of strain ZJ1T was 66.5 mol% (Tm). The stable phylogenetic position, differential physiological and biochemical properties and extensive sequence divergence suggest that strains ZJ1T and J81 represent a novel species, for which the name Halococcus salsus sp. nov. is proposed. The type strain is ZJ1T (=CGMCC 1.16025T=NBRC 112867T).

Halococcus agarilyticus sp. nov., an agar-degrading haloarchaeon isolated from commercial salt.

Two agar-degrading halophilic archaeal strains, 62 E(T) and 197 A, were isolated from commercial salt samples. Cells were non-motile cocci, approximately 1.2-2.0 µm in diameter and stained Gram-negative. Colonies were pink-pigmented. Strain 62 E(T) was able to grow with 24-30% (w/v) NaCl (optimum, 27%), at pH 6.5-8.5 (optimum, pH 7.5) and at 22-47 °C (optimum, 42 °C). The 16S rRNA gene sequences of strains 62 E(T) and 197 A were identical, and the level of DNA-DNA relatedness between them was 90 and 90% (reciprocally). The closest relative was Halococcus saccharolyticus JCM 8878(T) with 99.7% similarity in 16S rRNA orthologous gene sequences, followed by Halococcus salifodinae JCM 9578(T) (99.6%), while similarities with other species of the genus Halococcus were equal to or lower than 95.1%. The rpoB' gene tree strongly supported that the two strains were members of the genus Halococcus . Mean DNA-DNA relatedness between strain 62 E(T) and H. saccharolyticus JCM 8878(T) and H. salifodinae JCM 9578(T) was 46 and 44%, respectively. The major polar lipids were archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 archaeol, and sulfated diglycosyl archaeol-1. Several unidentified glycolipids were present. Based on the phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halococcus , for which the name Halococcus agarilyticus sp. nov. is proposed. The type strain is 62 E(T) ( = JCM 19592(T) =KCTC 4143(T)).

Adaptation, ecology, and evolution of the halophilic stromatolite archaeon Halococcus hamelinensis inferred through genome analyses.

Halococcus hamelinensis was the first archaeon isolated from stromatolites. These geomicrobial ecosystems are thought to be some of the earliest known on Earth, yet, despite their evolutionary significance, the role of Archaea in these systems is still not well understood. Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites. The genome of H. hamelinensis consisted of 3,133,046 base pairs with an average G+C content of 60.08% and contained 3,150 predicted coding sequences or ORFs, 2,196 (68.67%) of which were protein-coding genes with functional assignments and 954 (29.83%) of which were of unknown function. Codon usage of the H. hamelinensis genome was consistent with a highly acidic proteome, a major adaptive mechanism towards high salinity. Amino acid transport and metabolism, inorganic ion transport and metabolism, energy production and conversion, ribosomal structure, and unknown function COG genes were overrepresented. The genome of H. hamelinensis also revealed characteristics reflecting its survival in its extreme environment, including putative genes/pathways involved in osmoprotection, oxidative stress response, and UV damage repair. Finally, genome analyses indicated the presence of putative transposases as well as positive matches of genes of H. hamelinensis against various genomes of Bacteria, Archaea, and viruses, suggesting the potential for horizontal gene transfer.

Halophilic microorganisms are responsible for the rosy discolouration of saline environments in three historical buildings with mural paintings.

A number of mural paintings and building materials from monuments located in central and south Europe are characterized by the presence of an intriguing rosy discolouration phenomenon. Although some similarities were observed among the bacterial and archaeal microbiota detected in these monuments, their origin and nature is still unknown. In order to get a complete overview of this biodeterioration process, we investigated the microbial communities in saline environments causing the rosy discolouration of mural paintings in three Austrian historical buildings using a combination of culture-dependent and -independent techniques as well as microscopic techniques. The bacterial communities were dominated by halophilic members of Actinobacteria, mainly of the genus Rubrobacter. Representatives of the Archaea were also detected with the predominating genera Halobacterium, Halococcus and Halalkalicoccus. Furthermore, halophilic bacterial strains, mainly of the phylum Firmicutes, could be retrieved from two monuments using special culture media. Inoculation of building materials (limestone and gypsum plaster) with selected isolates reproduced the unaesthetic rosy effect and biodeterioration in the laboratory.

Halococcus sediminicola sp. nov., an extremely halophilic archaeon isolated from a marine sediment.

A novel, red-pigmented and coccoid haloarchaeon, designated strain CBA1101(T), was isolated from a marine sediment. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CBA1101(T) is most closely related to the genus Halococcus in the family Halobacteriaceae. Strain CBA1101(T) had a highest 16S rRNA gene sequence similarity of 98.4 % with Halococcus dombrowskii DSM 14522(T), followed by 93.7-98.3 % with sequences of other type strains in the genus Halococcus. The RNA polymerase subunit B' gene sequence similarity of strain CBA1101(T) with that of Halococcus qingdaonensis JCM 13587(T) is 89.5 % and lower with those of other members of the genus Halococcus. Strain CBA1101(T) was observed to grow at 25-40 °C, pH 6.0-9.0 and in the presence of 15-30 % (w/v) NaCl, with optimal growth at 35-40 °C, pH 7.0 and with 20 % NaCl. The cells of strain CBA1101(T) are Gram-negative and did not lyse in distilled water. The major polar lipids were identified as phosphatidylglyerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diether, unidentified phospholipids and unidentified glycolipids. The genomic DNA G+C content was determined 66.0 mol%. The DNA-DNA hybridization experiment showed that there was less than 40 % relatedness between strain CBA1101(T) and the reference species in the genus Halococcus. Based on this polyphasic taxonomic analysis, strain CBA1101(T) is considered to represent a new species in the genus Halococcus, for which the name Halococcus sediminicola sp. nov. is proposed. The type strain is CBA1101(T) (=JCM 18965(T) = CECT 8275(T)).

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species.

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and (1)H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307(T), Halococcus saccharolyticus DSM 5350(T), Halococcus salifodinae DSM 8989(T), Halococcus dombrowskii DSM 14522(T), Halococcus hamelinensis JCM 12892(T), Halococcus qingdaonensis JCM 13587(T)), Halorubrum sp. (Hrr. coriense DSM 10284(T), Halorubrum chaoviator DSM 19316(T), Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189(T), Natronococcus occultus DSM 3396(T)) and Halobacterium noricense DSM 9758(T). No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757(T). Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ss-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.

Occurrence of Halococcus spp. in the nostrils salt glands of the seabird Calonectris diomedea.

The nostrils of the seabird Calonectris diomedea are endowed with a salt-excreting gland that could produce a suitable environment for the colonization of extreme halophilic prokaryotes. We have studied in this organ the presence of extreme halophiles by means of culturing techniques. We could easily cultivate members of haloarchaea, and all cultures studied were identified as members of one of the two species Halococcus morrhuae and Hcc. dombrowskii. In order to reveal the diversity of these colonizers, we undertook a taxonomic study. Altogether, the results indicated that members of the genus Halococcus may constitute a part of the natural epizootic microbiota of C. diomedea, and that they exhibit such an important degree of taxonomic variability that appeals for a pragmatic species definition. This seabird nests in the west Mediterranean coasts, but its migratory habits, reaching locations as distant from the Mediterranean as the South Atlantic, may help in the dispersal mechanisms of haloarchaea through the Earth's surface.

Halococcus thailandensis sp. nov., from fish sauce in Thailand.

Fifteen strains of red-pigmented, strictly aerobic, coccoid, extremely halophilic archaea were isolated from fish sauce (nam-pla) produced in Thailand. They grew optimally at 37 degrees C, pH 6-8 and in the presence of 20-30 % (w/v) NaCl. The DNA G+C contents of the isolates were 60.0-61.8 mol%. They had MK-8(H2) as a major menaquinone component and C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol, phosphatidylglycerol methylphosphate and a sulfated glycolipid, S-DGA-1, as major polar lipid components. 16S rRNA gene sequence comparisons revealed that a representative strain, HDB5-2(T), was affiliated with Halococcus dombrowskii JCM 12289(T), Halococcus qingdaonensis JCM 13587(T) and Halococcus morrhuae JCM 8876(T) (levels of similarity of 98.2-98.7 %). Based on data from DNA-DNA hybridization experiments, the 15 strains represented a single species, showing hybridization values of >78.9 % to representative strain HDB5-2(T), but were unrelated to either Halococcus dombrowskii JCM 12289(T) or Halococcus morrhuae JCM 8876(T), with levels of relatedness of <50 %. Moreover, a comparison of phenotypic properties discriminated these new isolates from recognized species of the genus Halococcus. The 15 strains are thus considered to represent a novel species of the genus Halococcus, for which the name Halococcus thailandensis sp. nov. is proposed. The type strain is HDB5-2(T) (=BCC 20213(T) =JCM 13552(T) =PCU 278(T)).

Halococcus qingdaonensis sp. nov., a halophilic archaeon isolated from a crude sea-salt sample.

A Gram-negative, extremely halophilic, coccoid archaeal strain, CM5(T), was isolated from a crude sea-salt sample collected near Qingdao, China. The organism grew optimally at 35-40 degrees C and pH 6.0 in the presence of 20 % (w/v) NaCl. Its colonies were red in colour and it could use glucose as a sole carbon source for growth. The 16S rRNA gene sequence of CM5(T) was most closely related to those of Halococcus species. Its pattern of antibiotic susceptibility was similar to those of other described Halococcus species. Biochemical tests revealed no sign of H(2)S production or gelatin liquefaction. The main polar lipids of strain CM5(T) were phosphatidylglycerol, phosphatidylglycerol methylphosphate and sulfated diglycosyl diether. No phosphatidylglycerol sulfate was present. The DNA G+C content of strain CM5(T) was 61.2 mol% and it gave DNA-DNA reassociation values of 33.7, 57.1 and 29.6 %, respectively, with Halococcus salifodinae DSM 8989(T), Halococcus dombrowskii DSM 14522(T) and Halococcus morrhuae ATCC 17082(T). Based on its morphological and chemotaxonomic properties and phylogenetic analysis of 16S rRNA gene sequence data, we propose that CM5(T) should be classified within a novel species, Halococcus qingdaonensis sp. nov., with strain CM5(T) (=CGMCC 1.4243(T)=JCM 13587(T)) as the type strain.

Halococcus hamelinensis sp. nov., a novel halophilic archaeon isolated from stromatolites in Shark Bay, Australia.

Several halophilic archaea belonging to the genus Halococcus were isolated from stromatolites from Hamelin Pool, Shark Bay, Western Australia, collected during field trips in 1996 and 2002. This is the first incidence of halophilic archaea being isolated from this environment. Stromatolites are biosedimentary structures that have been formed throughout the earth's evolutionary history and have been preserved in the geological record for over 3 billion years. The stromatolites from Hamelin Pool, Western Australia, are the only known example of extant stromatolites forming in hypersaline coastal environments. Based on their 16S rRNA gene sequences and morphology, the isolates belong to the genus Halococcus. Strain 100NA1, isolated from stromatolites collected in 2002, was closely related to strain 100A6(T) that was isolated from the stromatolites collected in 1996, with a DNA-DNA hybridization value of 94 +/- 8 %. DNA-DNA hybridization values of strain 100A6(T) with Halococcus morrhuae NRC 16008 and Halococcus saccharolyticus ATCC 49257(T) were 17 +/- 6 and 11 +/-7 %, respectively. The DNA G + C content of strain 100A6(T) was 60.5 mol% (T(m)). The main polar lipid was S-DGA-1, a sulphated glycolipid that has been detected in all strains of the genus Halococcus. Whole-cell protein profiles, enzyme composition and utilization of various carbon sources were distinct from those of all previously characterized Halococcus species. The recognition of this strain as representing a novel species within the genus Halococcus is justified, and the name Halococcus hamelinensis sp. nov. is proposed. The type strain is 100A6(T) (=JCM 12892(T) = ACM 5227(T)).

Halococcus dombrowskii sp. nov., an archaeal isolate from a Permian alpine salt deposit.

Several extremely halophilic coccoid archaeal strains were isolated from pieces of dry rock salt that were obtained three days after blasting operations in an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225-280 million years ago). On the basis of their polar-lipid composition, 16S rRNA gene sequences, cell shape and growth characteristics, the isolates were assigned to the genus Halococcus. The DNA-DNA reassociation values of one isolate, strain H4T, were 35 and 38% with Halococcus salifodinae and Halococcus saccharolyticus, respectively, and 65.8-67.8% with Halococcus morrhuae. The polar lipids of strain H4T were C20-C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate. Whole-cell protein patterns, menaquinone content, enzyme composition, arrangements of cells, usage of carbon and energy sources, and antibiotic susceptibility were sufficiently different between strain H4T and H. morrhuae to warrant designation of strain H4T as a new species within the genus Halococcus. It is proposed that the isolate be named Halococcus dombrowskii, and the type strain is H4T (= DSM 14522T = NCIMB 13803T = ATCC BAA-364T).

Microorganismos halófitos: potencial biotecnológico e interés farmacéutico / Biotechnological potential and pharmaceutical interest of Halophilic Microorganisms

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Very similar strains of Halococcus salifodinae are found in geographically separated permo-triassic salt deposits.

The authors have previously isolated a novel extremely halophilic archaeon, Halococcus salifodinae Blp, from Austrian rock salt deposited about 250 million years ago. In this study they compared strain Blp with two other halococci isolated independently from geographically distant salt deposits of similar age, and with two recent isolates (N1 and H2) from the same site as strain Blp. Strain BG2/2 was from a salt mine in Germany and strain Br3 from a halite deposit in England; both resembled Hc. salifodinae Blp in cellular and colonial morphology. Strains Blp, BG2/2 and Br3 had identical 16S rRNA sequences, very similar whole-cell protein patterns, which were different from those of other halococci, similar G+C contents and identical sequences in a 108-base insertion in their 5S rRNA gene. Other similarities included composition and relative abundances of polar lipids, antibiotic susceptibility, enzymic activities and Fourier-transform infrared spectra. Strains N1 and H2 showed similar morphology, whole-cell protein patterns and biochemical characteristics as strains Blp, Br3 and BG2/2. Their partial 16S rRNA sequences (682 and 641 bases, respectively) were indistinguishable from those of strains Blp, Br3 and BG2/2. Therefore strains N1 and H2 can be considered as reisolates of Hc. salifodinae which were obtained 8 years after the first samples were taken from that mine. The results presented suggest that viable halophilic archaea, which belong to the same species, occur in widely separated evaporite locations of similar geological age, and support the notion that these halophilic isolates from subterranean salt deposits may be the remnants of populations which inhabited ancient hypersaline seas.