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.
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.Numerous archaea (archaebacteria) thrive in hostile conditions such as salt brines, hot springs, and acidic or alkaline environments (20). Their membrane lipids differ from those of (eu)bacteria and other organisms because they contain ether linkages instead of ester linkages, are composed of regularly branched phytanyl and biphytanyl chains instead of fatty acyl chains, and possess glycerol ethers, which are sn-2,3 substituted rather than sn-1,2 substituted (13). These properties are thought to contribute to the greater chemical stability of archaeal lipids (13,22) and the generally low permeability of archaeal membranes (5,14). The LIVE/DEAD BacLight bacterial viability kit (henceforth referred to as the LIVE/DEAD kit) from Molecular Probes is widely used for the enumeration of bacteria (2,8,12) and provides an indication of the fraction of active cells. The kit consists of two nucleic acid stains: SYTO 9, which penetrates most membranes freely, and propidium iodide, which is highly charged and normally does not permeate cells but does penetrate damaged membranes. Simultaneous application of both dyes therefore results in green fluorescence of viable cells with an intact membrane, whereas dead cells, because of a compromised membrane, show intense red fluorescence (10). Archaea have not been treated with the LIVE/DEAD kit, except for one psychrophilic isolate (15); in view of the low permeability of their membranes and their existence in habitats that often border on the physicochemical limits of life, it was of interest to determine if the LIVE/DEAD kit would detect extremophilic archaea and provide reliable information about their viability.Archaeal strains were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), except Halobacterium sp. strain NRC-1 ATCC 700922, which was obtained from LGC, London, United Kingdom. Haloarchaea were grown at 37°C in M2 medium (27), except Halococcus and haloalkaliphiles, which were grown in M2S medium (25) or Tindall's medium (26), respectively, and Halobacterium sp. strain NRC-1, which was grown in ATCC medium no. 2185 (http://www.lgcpromochem .com/atcc/). Acidianus brierleyi DSM 1651T (21) and Sulfolobus acidocaldarius DSM 639 T (31) were grown at 65 to 70°C in DSM medium no. 150 (http://www.dsmz.de/media/med150 .htm) and ATCC medium no. 1723 (http://www.atcc.org /SearchCatalogs/Search.cfm), respectively. The dyes of LIVE /DEAD BacLight kit L-7012 (Molecular Probes, Inc., Eugene, Oreg.) were freshly diluted with water and used as previously described (3, 10). Staining with 4Ј,6Ј-diamidino-2-phenylindole (DAPI) was done as described b...
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 1H-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 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
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.
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