Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters.

Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing hypotheses on prokaryotic evolution, such as the molecular clock concept, or the net-like history of genome evolution. A comparison of available taxonomic characteristics from strains of Hcc. salifodinae and other Halococcus species, most of them originating from surface waters, is presented. The cell wall polymer of Hcc. salifodinae was examined and found to be a heteropolysaccharide, similar to that of Hcc. morrhuae. Polyhydroxyalkanoate granules were present in Hcc. salifodinae, suggesting a possible lateral gene transfer before Permian times.

Responses of haloarchaea to simulated microgravity.

Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites).

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.

Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece.

Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2-97.1%. The DNA-DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75%, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2%, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5-66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T=NCIMB 14426T=ATCC BAA-1602T).

Extremely halophilic archaea and the issue of long-term microbial survival.

Halophilic archaebacteria (haloarchaea) thrive in environments with salt concentrations approaching saturation, such as natural brines, the Dead Sea, alkaline salt lakes and marine solar salterns; they have also been isolated from rock salt of great geological age (195-250 million years). An overview of their taxonomy, including novel isolates from rock salt, is presented here; in addition, some of their unique characteristics and physiological adaptations to environments of low water activity are reviewed. The issue of extreme long-term microbial survival is considered and its implications for the search for extraterrestrial life. The development of detection methods for subterranean haloarchaea, which might also be applicable to samples from future missions to space, is presented.

Evaluation of the LIVE/DEAD BacLight kit for detection of extremophilic archaea and visualization of microorganisms in environmental hypersaline samples.

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.

Halobacterium noricense sp. nov., an archaeal isolate from a bore core of an alpine Permian salt deposit, classification of Halobacterium sp. NRC-1 as a strain of H. salinarum and emended description of H. salinarum.

Two rod-shaped haloarchaeal strains, A1 and A2, were isolated from a bore core from a salt mine in Austria. The deposition of the salt is thought to have occurred during the Permian period (225-280 million years ago). The 16S rDNA sequences of the strains were 97.1% similar to that of the type species of the genus Halobacterium, which was also determined in this work. Polar lipids consisted of C20-C20 derivatives of phosphatidylglycerol, methylated phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and sulfated tetraglycosyl diether. Optimal salinity for growth was 15-17.5% NaCl; Mg++ was tolerated up to a concentration of 1 M. The DNA-DNA reassociation value of strain A1T was 25% with H. salinarum DSM 3754T and 41% with Halobacterium sp. NRC-1, respectively. Based on these results and other properties, e.g. whole cell protein patterns, menaquinone content and restriction patterns of DNA, strains A1 and A2 are members of a single species, for which we propose the name H. noricense. The type strain is A1 (DSM 15987T, ATCC BAA-852T, NCIMB 13967T). Since we present evidence that Halobacterium sp. NRC-1 is a member of H. salinarum, an emended description of H. salinarum is provided.

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).

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.