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Survival strategies for microorganisms in hypersaline environments and their relevance to life on early Mars.

There are two groups of microorganisms that live and grow in hypersaline (>10-15% NaCl) environments: the halophilic Archaea and the halotolerant Bacteria and algae. In order to grow and reproduce in such high-salt, low-water activity environments, these organisms have made basic biochemical adaptations in their proteins, osmoregulation mechanisms, nucleic acids, and lipids. The environment of the halophiles and especially how the halophilic Archaea have adapted to that environment are reviewed in this paper. Along with this review is a brief description of how these adaptations could be important in the detection of life on early Mars assuming similar types of salts and a carbon-based life.

Adaptation, Biological↗

A transcriptional reporter for in vivo promoter analysis in the archaeon Haloferax volcanii.

We have used a modified intron-containing tRNA(Pro(UGG) gene (tRNA(ProM), derived from the Saccharomyces cerevisiae tRNA(Pro(UGG) gene, as a reporter to measure in vivo transcription from a halophilic archaeon promoter. Coupling of the yeast tRNA(ProM) gene to the Haloferax volcanii tRNA(Lys) promoter on the H. volcanii plasmid pWL201 led to the production of a single stable transcript that was readily quantitated by Northern (RNA) blot analysis. Comparison of tRNA(ProM) RNA production from constructs containing the wild-type tRNA(Lys) promoter and those containing mutant tRNA(Lys) promoters demonstrated that this assay system can be used to measure expression from strong and weak promoters.

Base Sequence↗

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.

Genetic Vectors↗

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii.

The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker.

Amino Acid Sequence↗

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.

Biological Evolution↗

Identification of sulfoquinovosyl diacylglycerol as a major polar lipid in Marinococcus halophilus and Salinicoccus hispanicus and substitution with phosphatidylglycerol.

The sulfonolipid sulfoquinovosyl diacylglycerol normally associated with photosynthetic membranes was identified as a major lipid in Marinococcus halophilus, Salinicoccus hispanicus ("Marinococcus hispanicus"), and Marinococcus sp. H8 (Planococcus sp. H8). Phosphatidylglycerol and 0%-10% cardiolipin accounted for the remaining polar lipids in these moderately halophilic, Gram-positive bacteria. Negative-ion fast atom bombardment mass spectrometry was used to quantify these three polar lipids from cells grown in media containing 0.03 to 4 mol NaCl/L. All strains revealed dramatic shifts in the ratio of sulfonolipid to phospholipid dependent on the salinity of the growth media, when grown in media with low phosphate content. Highest sulfonolipid content occurred during best growth in 0.5-2 mol NaCl/L, approaching 80%-90% of the total polar lipids. It was demonstrated that growth of M. halophilus in the presence of elevated phosphate and low sulfate blocked the shift to decreased phospholipids most notably during growth in 0.5-2 mol NaCl/L, without significant influence on growth. The data suggest that in low-phosphate media the influence of NaCl concentration on growth rate (and resulting demand for phosphate by competing pathways) is the primary factor responsible for exchange between phospholipid and sulfonolipid. We conclude that sulfoquinovosyl diacylglycerol, by substitution with phospholipids, contributes to the ability of these Gram-positive cocci to adapt to changing ionic environments. A comparison of 16S rRNA established a close similarity between Planococcus sp. H8 and M. halophilus.

Glycolipids↗

Organic solvent tolerance of halophilic archaea, Haloarcula strains: effects of NaCl concentration on the tolerance and polar lipid composition.

Strains of halophilic archaea, Haloarcula vallismortis and two Haloarcula strains OHF-1 and OHF-2, showed high tolerance to organic solvents at high media NaCl concentrations. For example, the lowest log Pow of the solvent which allowed growth (log Pow is the common logarithm of the partition coefficient of a given solvent in a mixture of n-octanol and water) for H. vallismortis was 5.1 at 20% NaCl and 4.4 at 30% NaCl. The solvent tolerance of Haloarcula argentinensis, on the other hand, was not affected by the NaCl concentration. Cells of strains OHF-1 and OHF-2 were of triangular or irregular morphology but became spherical in cultures in NaCl media overlaid with cyclohexane (log Pow=3.4), but returned to the triangular shape when the organic solvent evaporated from the medium. When cells of strains OHF-1, OHF-2, and H. argentinensis were grown in NaCl media in the presence of n-decane, they contained less phosphatidylglycerol and more phosphatidylglycerosulfate and phosphatidylglycerophosphate methyl ester than when grown without added n-decane. When the solvent was removed from the media after cultivation, the levels of these compounds returned to their initial ones.

Cell Proliferation↗

Biotechnological potential of halobacteria.

The extremely halophilic archaebacteria (halobacteria) became an early focus of scientific interest owing to their role in salted food deterioration. In more recent times their peculiar physiology involving extreme adaptation to the salt environment and other unique features have allowed the development of other applied interests. Their similarities to eukaryotic cells at the level of cell division justifies their use in the prescreening for anti-cancer drugs, and some of their antigens could be used for cancer diagnosis. Their unique retinal proteins can be used as light-biosensors and the use of the purple membrane (pm) as reversible holographic medium has already been developed. Halobacterial enzymes are an extremely tough raw material for enzyme technology, particularly for applications in which the reaction mixture has very low water activity. Thanks to their peculiar lipids and to the production of polysaccharides by some halobacteria, their cultures could be used for enhanced oil recovery. Some halobacteria are excellent producers of industrially interesting biopolymers. The use of halobacteria as producers of polyhydroxyalkanoates, biological polyesters such as poly-3-hydroxybutyrate, with the properties of biodegradable thermoplastics, is being considered.

Biotechnology↗

[Diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China].

The aims of this work were to explore the diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China and to collect novel halophilic archaea. One hundred and sixty-five halophilic archaea were isolated from the three different types of hypersaline lakes (Erliannor, shangmatala and Xilin soda lake) in Inner Mongolia. By analysis of the restriction patterns of amplified 16S rDNA (ARDRA) with the enzyme Afa I and Hae II, respectively, the isolates were clustered into 14 genotypes, and the representatives of each genotype were randomly chosen for the determination of 16S rDNA sequence. The phylogenetic analysis revealed that all of the isolates were clustered into 10 groups: Halorubrum, Natronococcus, Natronorubrum, Haloterrigena, Halorhabdus, Halobiforma, Haloarcula, Haloferax and other two unknown groups. Dominant isolates were related to Halorubrum spp. in all three lakes. Some of the isolates studied showed less affiliation with known taxa ( <98% sequence similarity) and may represent novel taxa. Two isolates HXH33 and HSH33 showed very less affiliation with known genus ( < 93% sequence similarity) and may represent two new genera. These results suggest that diverse archaea exist in and the unknown archaea thrive in the hypersaline lakes of Inner Mongolia.

Biodiversity↗

[Adaptation strategies of halophilic microorganisms and Debaryomyces hansenii (halophilic yeast)].

The term halophile is used for all those organisms belonging to hypersaline habitats; they constitute an interesting class of organisms able to compete successfully in salt water and to resist its denaturing effects. A wide diversity of microorganisms, prokaryotic and eukaryotic belong to this category. Halophile organisms have strategies allowing them not only to withstand osmotic stress, but also to function better in the presence of salt, in spite of maintaining high intracellular concentrations of salt, partly due to the synthesis of compatible solutes that allow them to balance their osmotic pressure. We describe the characteristics of some halophile organisms and D. hansenii (halophile yeast), that allow them to resist high concentrations of salt. The interest to know the great diversity microorganisms living in hypersaline habitats is growing, and has begun to be the center of recent investigations, since halophile organisms produce an wide variety of biomolecules that can be used for different applications. In this review we describe some mechanisms with which some halophile organisms count to resist the high concentration of salts, mainly NaCl.

Adaptation, Physiological↗

Optimization of the production of a bacteriocin from Haloferax mediterranei Xia3.

The optimal conditions for the production of the halocin H1, a 31 kDa bacteriocin-like molecule produced by the extreme halophilic Archaea Haloferax mediterranei Xia3 active against Gram-negative haloarchaea, was characterized. The physico-chemical conditions required for the optimal production of halocin H1 are similar to those found in the habitat in which the microorganism was isolated: 20% salt concentration and temperature range between 37 and 42 degrees C. Optimal antimicrobial activity was obtained using 0.5% of N-Z amine E as nutrient.

Bacteriocins↗

[The role of halophilic bacteria in decarboxylation of histidine in salted fish].

The purpose of this study was to determine frequency of occurrence of halotolerant and halophilic histamine-forming bacteria under laboratory conditions in salted herring stored at 20 degrees C for 2 months. The other aim was to isolate these bacteria both from herring bought in a retail shop and examine the ability of decarboxylation of histidine by these microorganisms using Karnop's method modified by the authors. The amount of histamine formed was determined by fluorometric method. The level of histamine in media containing homogenates of herring was in a range from 5 to 1950 micrograms/100 ml of a nutrient medium. All isolated bacteria belong to halotolerant and halophilic strains and produce histamine both at 8% and 20% content of NaCl in a nutrient medium. The obtained results indicate that during storage of salted herring at ambient temperature the increase of histamine content in their flesh can occur.

Animals↗