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Halobacteria: the evidence for longevity.

Subterranean salt deposits are the remains of ancient hypersaline waters that presumably supported dense populations of halophilic microorganisms including representatives of the haloarchaea (halobacteria). Ancient subterranean salt deposits (evaporites) are common throughout the world, and the majority sampled to date appear to support diverse populations of halobacteria. The inaccessibility of deep subsurface deposits, and the special requirements of these organisms for survival, make contamination by halobacteria from surface sites unlikely. It is conceivable that these subterranean halobacteria are autochthonous, presumably relict populations derived from ancient hypersaline seas that have been revived from a state of dormancy. One would predict that halobacteria that have been insulated and isolated inside ancient evaporites would be different from comparable bacteria from surface environments, and that it might be possible to use a molecular chronometer to establish if the evolutionary position of the subsurface isolates correlated with the geological age of the evaporite. Extensive comparisons have been made between the 16S rRNA genes of surface and subsurface halobacteria without showing any conclusive differences between the two groups. A further phylogenetic comparison exploits an unusual feature of one particular group of halobacteria that possess at least two heterogeneous copies of the 16S rRNA gene, the sequences of which may have been converging or diverging over geological time. However, results to date have yet to show any gene sequence differences between surface and evaporite-derived halobacteria that might arguably be an indication of long-term dormancy.

Evolution, Molecular↗

Moderately halophilic gram-positive bacterial diversity in hypersaline environments.

Moderately halophilic bacteria are microorganisms that grow optimally in media containing 3%-15% (w/v) salt. They are represented by a heterogeneous group of microorganisms included in many different genera. Gram-negative moderately halophilic bacteria have been studied in more detail, but studies on gram-positive species are more scarce. Recent studies carried out by our research group on gram-positive moderate halophiles have permitted clarifying their taxonomic and phylogenetic position and describing new species. Thus, we have isolated six strains from ponds of salterns that show phenotypic and genotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius), a moderately halophilic gram-positive coccus that was described on the basis of a single strain. Our data demonstrate quite clearly that they are members of this species and contribute to a better description of these moderately halophilic cocci. Similarly, a study of a large number of gram-positive moderately halophilic rods that were able to produce endospores led us to describe a new species, designated Bacillus salexigens. Further, isolates grouped in other three phenons, obtained by numerical taxonomy analysis and showing phenotypic features quite similar to those of this species, represent different genomovars, with very low DNA-DNA homology. Although they might represent additional new species, it will be necessary to determine new phenotypic features to differentiate them from previously described Bacillus species. We have also studied the viability of some old enrichments provided by B.E. Volcani, which were set up in 1936. We isolated 31 gram-positive motile endospore-forming rods that, according to their phenotypic characteristics, could represent a new species of the genus Bacillus.

Bacillus↗

Distribution and diversity of halophilic bacteria in a subsurface salt formation.

The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy. The facility will be used for permanent disposal of transuranic wastes. This underground repository has been constructed in the geologically stable Permian age Salado salt formation. Of the wastes to be placed into the facility, 85% will be biodegradable cellulose. A 3-year survey of the bacterial populations existing within the facility was conducted. Bacterial populations were found to be heterogeneously distributed throughout the mine. Populations in some mine areas reached as high as 1.0 x 10(4) colony-forming units per gram of NaCl. The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity. A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups. The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds. When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation. Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids. The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations.

Acids↗

Halophilic adaptation of enzymes.

It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly.

Adaptation, Physiological↗

Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, USA, and Eilat, Israel.

The whole community pigments and lipids have been examined during a 5-year period in two commercial solar salterns located in the United States and in Israel. There were significant differences in the complexity of the lipid and pigment patterns within the California saltern system, and these differences were not consistent over the sampling period despite examination of ponds with the same salinity. The solar saltern system in Eilat, Israel, showed greater consistency during this sampling period and compared directly with previous studies. The complexity of the saltern in Newark, California, could be explained on the basis of the prevailing weather conditions (cooler and more rainfall) and the nutrient-enriched source water. The Eilat saltern, however, has an oligotrophic water source and has a considerably warmer and drier climate. This difference resulted in more diverse and more complex pigment and lipid patterns and presumably microbial populations in the Newark, California, plant than in the saltern in Eilat, Israel.

Biomass↗

Extracellular protease of Natrialba magadii: purification and biochemical characterization.

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.

Amino Acid Sequence↗

Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins.

Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.

Amino Acids↗

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii.

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.

Archaeal Proteins↗

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

Cell Membrane↗

The N-terminal sequence of the ribosomal 'A' protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 11227).

The 'A' protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The 'A' protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the 'A' proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the 'A' protein from both moderate halophiles showed substantial homology to EL 12 (75--80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli were also isolated and their amino acid compositions determined.

Amino Acid Sequence↗

Phototactic behaviour of the archaebacterial Natronobacterium pharaonis.

Natronobacterium pharaonis can react tactically to photo- and chemostimuli. It moves by rotation of a flagellar bundle which is monopolarly inserted. Under sufficient oxygen supply the photophobic response of N. pharaonis has been measured. The resulting action spectrum matches the absorption spectrum of the purified retinylidene protein psR-II. Retical synthesis could be inhibited by nicotine. Cells grown in the presence of nicotine show a strongly reduced photoresponse, which could be restored by addition of retinal. These data identify psR-II as the receptor for negative phototaxis.

Chemotaxis↗

A novel prenyltransferase, farnesylgeranyl diphosphate synthase, from the haloalkaliphilic archaeon, Natronobacterium pharaonis.

A novel prenyltransferase, farnesylgeranyl diphosphate (FGPP) synthase (EC 2.5.1.X), which synthesizes C25-prenyl diphosphate, was found in the haloalkaliphilic archaeon Natronobacterium pharaonis. It was separated from geranylgeranyl diphosphate (GGPP) synthase (EC 2.5.1.29), which synthesizes C20-prenyl diphosphate, a major prenyltransferase in this organism. The highest activity of FGPP synthase was observed when GGPP was used as the allylic substrate. FGPP synthase may synthesize a precursor for the C25 moiety of C20, C25 diether lipids using a longer allylic diphosphate, such as GGPP synthesized by GGPP synthase, rather than dimethylallyl diphosphate, which is the product of isopentenyl diphosphate isomerase.

Alkyl and Aryl Transferases↗

Three different but related gene clusters encoding gas vesicles in halophilic archaea.

We present an analysis of the chromosomal region comprising the gene cluster involved in gas vesicle (Vac) synthesis in Haloferax mediterranei (mc-vac-region) and Halobacterium salinarium (c-vac-region) and compare both of them to the plasmid located p-vac-region of H. salinarium. The p-vac-region of 9000 base-pairs (9 kb) is more related to mc-vac (9.4 kb) of Hf. mediterranei than it is to the c-vac-region (8.3 kb) present in the same cell. The Vac- species Hf. volcanii becomes Vac+ following transformation with a fragment containing the entire mc-vac-region. Also the p-vac-region transforms Hf. volcanii to a Vac+ phenotype, indicating that this gene cluster is sufficient for gas vesicle synthesis and does not depend on products of the c-vac-region. Each of these vac-regions contains, in addition to gvpA encoding the major gas vesicle protein, 13 open reading frames named gvpC through gvpO. Ten of these, gvpD through gvpM, are located upstream from gvpA in opposite orientation, while gvpC, gvpN and gvpO are found 3' to gvpA. The absolute requirement of gvpO for gas vesicle synthesis was demonstrated by transformation experiments. Northern analyses with RNA samples isolated during the growth cycle of Hf. mediterranei or of H. salinarium PHH4 revealed that the mc-gvpD or c-gvpD mRNAs occur similar to the respective gvpA mRNA in stationary growth phase, while gvpF-gvpM are transcribed mainly during logarithmic growth. S1-nuclease mapping was performed to determine the transcriptional start site of the gvpD mRNA. The distance between the two divergent start sites of gvpA and gvpD mRNA is 109 base-pairs in mc-vac and p-vac, while in the case of c-vac this distance is 22 base-pairs larger. The conservation of the various gvp products, characteristic features and their possible functions in gas vesicle synthesis are discussed.

Archaeal Proteins↗

HL35e and HLA: primary structure of two very basic and cysteine-rich ribosomal proteins from Haloarcula marismortui.

Two small and very basic ribosomal proteins have been purified from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui by RP-HPLC. The complete primary structures of these two proteins, which we refer to as HL35e and HLA, have been determined by protein chemical methods. Both proteins are characterized by a high content of basic amino acids and the presence of two pairs of cysteines in each polypeptide chain, one of which resembles the C4-zinc-finger motif. Comparison of the protein sequences with those of other ribosomal proteins revealed that HL35e shows significant sequence homology exclusively to eukaryotic ribosomal proteins, namely to yeast L35 and to L37 from rat. For HLA no homologous ribosomal protein so far known could be found. Obviously, HL35e and HLA have no counterparts in eubacterial ribosomes.

Amino Acid Sequence↗

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei.

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36 degrees C in the presence of 1.5 M of (NH4)2SO4, 100 mM of MgAc2, 20 mM Tris-HCl (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K+ and NH4+ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg2+ concentration present in the system.

Halobacteriaceae↗

Survival strategies of microorganisms in extreme saline environments.

Halophilic representatives are found in all main lines of evolutionary descendence of microbes: in archaebacteria, Gram-negative and Gram-positive eubacteria, and also in eucaryotes. In principle all halophilic microorganisms have to adapt their surface and membrane structures to their highly ionic environments. Concerning their intracellular compartment two different strategies have been developed: Inorganic ions are largely excluded in some microorganisms while such ions are actively accumulated in others. In particular the second group of organisms has to adapt the whole metabolic machinery to the highly ionic conditions of several molar salts, whereas in the first group only the outer surface of the cytoplasmic membrane and the extracytoplasmic structures are in contact with high concentrations of inorgainic ions. In this latter group, a variety of organic solutes is accumulated in response to increases of the salinity of the environment.

Adaptation, Physiological↗

Crystallography of ribosomes: attempts at decorating the ribosomal surface.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Bacteria↗