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1.  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.
doi:10.1007/s11157-006-0007-y
PMCID: PMC3188376  PMID: 21984879
Extreme halophiles; Haloarchaea; Life detection; Microbial longevity; Salt mines; Salt sediments; Space missions; Subterranean; Taxonomy of halobacteriaceae
2.  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).
doi:10.1099/ijs.0.000463-0
PMCID: PMC3182535  PMID: 19567575
3.  Responses of Haloarchaea to Simulated Microgravity 
Astrobiology  2011;11(3):199-205.
Abstract
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). Key Words: Haloferax mediterranei—Halococcus dombrowskii—Simulated microgravity—Rotary cell culture system—Antibiotic resistance—Lithopanspermia. Astrobiology 11, 199–205.
doi:10.1089/ast.2010.0536
PMCID: PMC3079168  PMID: 21417742
4.  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 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 material
The online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-010-2611-6
PMCID: PMC2895300  PMID: 20437233
Polyhydroxybutyrate; Haloarchaea; Halococcus; Halobacterium; Haloalkaliphile
5.  Evaluation of the LIVE/DEAD BacLight Kit for Detection of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples 
Applied and Environmental Microbiology  2004;70(11):6884-6886.
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.
doi:10.1128/AEM.70.11.6884-6886.2004
PMCID: PMC525124  PMID: 15528557

Results 1-5 (5)