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...

Evidence for the widespread occurrence of extraterrestrial halite, particularly on Mars, has led to speculations on the possibility of halophilic microbial forms of life; these ideas have been strengthened by reports of viable haloarchaea from sediments of geological age (millions of years). Raman spectroscopy, being a sensitive detection method for future astrobiological investigations onsite, has been used in the current study for the detection of nine different extremely halophilic archaeal strains which had been embedded in laboratory-made halite crystals in order to simulate evaporitic conditions. The cells accumulated preferentially in tiny fluid inclusions, in simulation of the precipitation of salt in natural brines. FT-Raman spectroscopy using laser excitation at 1064 nm and dispersive micro Raman spectroscopy at 514.5 nm were applied. The spectra showed prominent peaks at 1507, 1152 and 1002 cm −1 which are attributed to haloarchaeal C 50 carotenoid compounds (mainly bacterioruberins). Their intensity varied from strain to strain at 1064-nm laser excitation. Other distinguishable features were peaks due to peptide bonds (amide I, amide III) and to nucleic acids. No evidence for fatty acids was detected, consistent with their general absence in all archaea.These results contribute to a growing database on Raman spectra of terrestrial microorganisms from hypersaline environments and highlight the influence of the different macromolecular composition of diverse strains on these spectra.

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.

The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522(T) was exposed to UV doses over a wavelength range of 200-400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m(2), and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m(2). The estimated D(37) (dose of 37 % survival) for Hcc. dombrowskii was > or = 400 kJ/m(2). However, exposure of cells to UV flux while in liquid culture reduced D(37) by 2 orders of magnitude (to about 1 kJ/m(2)); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.

Viable extremely halophilic archaea (haloarchaea) have been isolated from million‐year‐old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000‐ to 34 000‐year‐old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod‐shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of aw ≤ 0.75, but exposure to buffered 4 m LiCl (aw ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re‐grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50‐fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.