Detection methods for halite-embedded haloarchaea with potential relevance for extraterrestrial samples

Halophilic archaebacteria (haloarchaea) are found in environments with salt concentrations approaching saturation, such as natural brines, marine solar salterns and alkaline salt lakes; they have also been isolated from ancient subsurface salt sediments of great geological age (195–280 million years). Some of the strains from geologically old sediments were formally classified and described as novel species [1]. The characterization of subsurface microbial life is of astrobiological interest since extraterrestrial halite has been detected in meteorites and also on Mars. It is also conceivable that, due to the harsh surface conditions on Mars, microbial life, if existent, may have retreated into the subsurface. The renewed ExoMars plans of ESA and NASA call for an orbiter and two rovers, one of which will carry analytical instruments dedicated to exobiology and geochemistry research, including a subsurface drill [5]. Methods for the detection of microorganisms from subsurface locations are being developed; we are focussing on the detection of haloarchaea within natural or artificial halite samples. Three types of procedures were investigated here: Raman spectroscopy, staining of cells with fluorescent dyes, which provide also information on viability, and microarrays with a mixture of 300 antibodies against microbial cell components [4]. Raman spectroscopy is considered a very sensitive detection method for future astrobiological investigations on site. Its use for the detection of nine different extremely halophilic archaeal strains which had been embedded in laboratory-made halite crystals was explored [2]. The cells accumulated preferentially in tiny fluid inclusions of halite, as was demonstrated by pre-staining with the fluorescent green dye SYTO 9 (see Fig. 1). FTRaman spectroscopy with laser excitation at 1064 nm yielded spectra with prominent peaks at 1507, 1152 and 1002 cm, which were attributed to haloarchaeal C50 carotenoid compounds (mainly bacterioruberins) and whose intensity varied from strain to strain. Analysis of cells following embedding in halite was based on the labeling with fluorescent dyes which are contained in the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes). The kit consists of two nucleic acid stains: SYTO 9, which penetrates most membranes freely, and propidium iodide, which is highly charged and normally cellimpermeant; it will, however, penetrate damaged membranes. Green fluorescence thus indicates viable cells with an intact membrane, whereas dead cells, due to a compromised membrane, show red fluorescence. A related method was staining of cells with the membrane potential kit, also from Molecular Probes, which provides information on the presence or absence of a membrane potential. Antibody microarrays are immunosensors capable of detecting many different compounds from microbial species in complex mixtures such as environmental samples [4]. Positive signals were obtained with antibody mixtures and samples from Mediterranean sea salt or from the Dead Sea, which contained halophilic archaea and bacteria.