Extreme Halophilic Bacteria Research Articles

Extreme halophilic bacteria promote the surface dolomitization of calcite crystals in solutions with various magnesium concentrations

In this study, we conducted experiments on the transformation from calcite to protodolomite using bacteria. The surface dolomitization of calcite seed crystals was initiated by Vibrio harveyi J4, a newly isolated extreme halophilic bacterium, under increasing magnesium concentrations of 0, 0.05, 0.10, 0.15, to 0.20 mol L–1. The mineralogy, surface morphology, elemental composition, and lattice structure of the calcite crystals before and after bacterial treatment were analyzed by XRD, SEM-EDS, AFM and TEM, respectively. Organic functional groups within newly-formed minerals were determined by FTIR and XPS. The weight loss of minerals in the experimental group and control groups was measured by TGA and DSC. The experiments resulted in numerous nano-spheroidal-hemispheroidal precipitates being formed on the surface of the calcite seeds, whereas dissolution took place on the calcite crystal surfaces in control experiments with no bacteria. The nano-spheroids-hemispheroids were confirmed to be disordered protodolomite. The number of protodolomite spheroids on the calcite crystal surfaces increases dramatically with increasing concentration of magnesium. Since no calcium ions were added to the bioreactor, the protodolomite nano-spheroids-hemispheroids are considered to have been formed through some dissolution of the calcite to provide the Ca2+ ions in a local precipitation process. The mechanism of protodolomite precipitation is thought to be related to the presence of acidic amino acids and polysaccharides in the EPS of the extreme halophilic bacteria. These results suggest that in addition to promoting the precipitation of dolomite, micro-organisms may also play a key role in the dolomitization of calcite.

High Mg/Ca Molar Ratios Promote Protodolomite Precipitation Induced by the Extreme Halophilic Bacterium Vibrio harveyi QPL2.

Bacterial activities have been demonstrated as critical for protodolomite precipitation in specific aqueous conditions, whereas the relationship between the various hydrochemical factors and bacterial activity has not been fully explored. In this study, biomineralization experiments were conducted using a newly isolated extreme halophilic bacterium from salina mud, Vibrio harveyi QPL2, under various Mg/Ca molar ratios (0, 3, 6, 10, and 12) and a salinity of 200‰. The mineral phases, elemental composition, morphology, and crystal lattice structure of the precipitates were analyzed by XRD, SEM, and HRTEM, respectively. The organic weight and functional groups in the biominerals were identified by TG-DSC, FTIR, and XPS analysis. The amounts of amino acids and polysaccharides in the EPS of QPL2 cultured at various Mg/Ca molar ratios were quantified by an amino acid analyzer and high-performance liquid chromatography. The results confirm that disordered stoichiometric protodolomite was successfully precipitated through the activities of bacteria in a medium with relatively high Mg/Ca molar ratios (10 and 12) but it was not identified in cultures with lower Mg/Ca molar ratios (0, 3, and 6). That bacterial activity is critical for protodolomite formation as shown by the significant bacterial relicts identified in the precipitated spherulite crystals, including pinhole structures, a mineral coating around cells, and high organic matter content within the crystals. It was also confirmed that the high Mg/Ca molar ratio affects the composition of the organic components in the bacterial EPS, leading to the precipitation of the protodolomite. Specifically, not only the total EPS amount, but also other facilitators including the acidic amino acids (Glu and Asp) and polysaccharides in the EPS, increased significantly under the high Mg/Ca molar ratios. Combined with previous studies, the present findings suggest a clear link between high Mg/Ca molar ratios and the formation of protodolomite through halophilic bacterial activity.

极端嗜盐菌筛选鉴定及诱导碳酸盐矿化研究

极端嗜盐菌筛选鉴定及诱导碳酸盐矿化研究

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Isolation and identification of halophilic and halotolerant bacteria from the sediments of the Qeshm Island mangrove forest

Due to specific environmental and ecological conditions, mangrove forests are known as marine transitional zones between sea and land, and, as such, they host organisms with high ecological plasticity. The mangrove forests of Qeshm Island (Iran) are relatively pristine habitats and represent an ideal target for investigating patterns of either aquatic or benthic biodiversity. To provide insights on microbial diversity in this area, nineteen halophilic and halotolerant bacteria were isolated from the sediments in 2017 during low tide. The extracted bacterial strains were studied morphologically by streaking, initial observation of colonies and bacterial staining, and characterized using a battery of biochemical tests including KOH, MR, VP, urease, TSI, S/I/M, Mac, LIA, ODC, ADH, oxidase, catalase, and tryptophan deaminase. The optimum growth of halophilic bacteria was observed in salt concentrations from 5 to 20% NaCl, whereas the extreme halophilic Gram-positive strain grew in salt concentration of up to 30% NaCl. Molecular analyses were also carried out on four halophilic strains and one extreme halophilic gram-positive bacteria. Phylogenetic taxonomy analysis, after 16S rDNA gene Sanger sequencing, revealed that the halophilic bacteria were closely related to the strain types of the genus Bacillus including Bacillus licheniformis, Bacillus velezensis, Bacillus Paralicheniformis and Bacillus sp. with 99% bootstrap value. The extreme halophilic strain was associated to strains of Planococcus plakortidis with 100% bootstrap value.

Halophilic Bacteria Species in Fishery Products and Industrial Applications

Su ürünlerine tuz ilave edilmesi ürünlerin su aktivitesini düşürmekte bozulmayı geciktirmektedir. Ancak tuzlanmış, salamura, marine edilmiş, fermente su ürünlerinde tuzu seven ve tuza tolerans gösteren halofilik bakterilerin gelişimine neden olabilmektedir. Bu nedenle işlenmiş su ürünlerinde bozulmaya daha çok tuzu seven ve tuza tolerans gösteren halofilik bakteriler sebep olmaktadır. Halofilik bakterilerin işlenmiş su ürünlerinde gelişimi kalite kaybına neden olabilmektedir ancak tuza toleranslı olan bazı halofilik bakterileri türlerinin fermente ürünlerde gelişimi ürünlerde istenen tat ve aroma gelişimine neden olabilmektedir. Ayrıca halofilik bakterilerin endüstriyel alanda çok sayıda kullanım alanıda bulunmaktadır. Bu nedenle bu derleme çalışmasında halofilik bakterileri türleri, denizlerden, tuz göllerinden, tuzlardan, tuzlanmış ve fermente balık ürünlerinden izole edilen halofilik bakteriler, tuz ve tuzlanmış ve fermente balık ürünleri tüketiminin insan sağlığına etkileri, su ürünlerinde halofilik bakterilerin inhibisyonu ve halofilik bakterilerin endüstriyel alanda kullanımları konularında yapılmış çalışmalara değinilmiştir.

Enhanced Production and Characterization of a Highly Stable Extracellular Protease from an Extreme Halophilic Isolate Salicola marasensis.

Owing to their superior catalytic activity in the extreme conditions, extremozymes have found the potential biotechnological applications for industrial purposes. A robust extracellular protease activity was detected in the culture broth of Salicola marasensis, an extreme halophilic bacterium, after a 48 h-incubation. The effect of different media ingredients in a liquid state fermentation was followed with the aim of improving the enzyme production yield. Fractional factorial and Box-Behnken designs were applied to get a 3.4 fold (from 6.0 to 20.3 U mL-1) improvement of protease production. The distinguishing features of this enzyme were stability at a wide range of pH (5.0-11.0) and temperature (25-60 °C), significant compatibility towards organic solvents, metal ions, chemicals, and surfactants, and hydrolysis of a variety of substrates. The properties of this enzyme can be of tremendous help in terms of the halophilic proteolytic extract's industrial applications.

Characterization of Some Extremely Halophilic Bacteria Isolated from Salt Marshes of Gamasa Egypt

Halophiles can be classified as extreme, moderate and slight halophiles. Many extremophiles produce unique biotechnological and commercial materials. Salinity stress is one of the major factors negatively affecting bacterial growth. Cellular biochemical changes could be monitored via protein pattern in response to different NaCl concentrations. So our work aimed to do a comparison among salt-tolerant bacteria in their response to different NaCl concentrations via halotolerance test, antimicrobial susceptibility, amino acid profile and protein banding pattern. From haloterance test of eleven bacterial isolates, STB5, STB8 and Shigella dysenteriae were more tolerance to NaCl. From 16S rRNA gene sequencing, the two isolates STB5 and STB8 were identified as Halomonas caseinilytica and Paraliobacillus quinghaiensis. From 32used antibiotics only 5 antibiotics, cefotaxime, ampicillin, penicillin G, ceftazidime and aztreonam, showed resistance. From SDS PAGE, the appearance or absence of high or low molecular weight bands may be directly associated with the bacterial response to salt. Protein profile of tested halophilic bacteria under different concentrations of NaCl% (0, 5% and 20%) showed variable polymorphism in Halomonas caseinilytica MG199079, Paraliobacillus quinghaiensis MG250199and Shigella dysenteriae. Environment-specific patterns of amino acids profile were observed. Based on the obtained results, it could be concluded that the three organisms Halomonas caseinilytica MG199079, Paraliobacillus quinghaiensis MG250199and Shigella dysenteriae, are extreme halophilic bacteria, they were different in of amino acids accumulation protein profile and in different concentration of NaCl.

Erratum for Behera et al., Draft Genome Sequence of the Extremely Halophilic Bacterium Halomonas salina Strain CIFRI1, Isolated from the East Coast of India.

Volume 3, no. 1, [e01321-14][1], 2015. Page 1: The affiliation line should read as given above. [1]: /lookup/doi/10.1128/genomeA.01321-14

Draft Genome Sequence of the Extremely Halophilic Bacterium Halomonas salina Strain CIFRI1, Isolated from the East Coast of India.

Halomonas salina strain CIFRI1 is an extremely salt-stress-tolerant bacterium isolated from the salt crystals of the east coast of India. Here we report the annotated 3.45-Mb draft genome sequence of strain CIFRI1 having 86 contigs with 3,139 protein coding loci, including 62 RNA genes.

Effect of Complex Nutrients and Repeated-Batch Cultivation of Halobacterium salinarum on enhancing Bacteriorhodopsin Production

The extreme halophilic bacteria Halobacterium salinarum is known to produce bacteriorhodopsin (BR) protein in its purple membrane (PM) as a light-driven pump for the synthesis ATP. Its growth does not utilize simple carbon sources such as glucose, but relies on the complex carbon/nitrogen sources. The production yield of BR in the culture of H. salinarum also strongly depends on the complex nitrogen sources employed. From the various complex carbon/nitrogen sources employed in this work, tryptone rather than commonly used peptone was found to be the best complex nutrient for the growth of H. salinarum and its BR production. Bubble column photobioreactor operated in a repeated-batch mode was also employed to enhance the growth of H. salinarum by intermittent removing growth inhibitory metabolites. By employing 0.5% tryptone as carbon/nitrogen source in the culture medium, 201.8 mg/L of BR was obtained after 210 h repeated-batch cultivation which is about 50% higher than that obtained in shaker flask cultivation.

Biosynthesis and characterization of polyhydroxyalkanoates produced by an extreme halophilic bacterium, Halomonas nitroreducens, isolated from hypersaline ponds.

Morphological, biochemical and genotypic characterization of a halophilic bacterium isolated from hypersaline ponds located at Las Coloradas (Río Lagartos, Yucatán, Mexico). Characterization of polymer produced by this strain was also performed. Twenty strains were isolated from water samples of salt ponds and selected based on both morphological features and their PHA storage capacity, which were determined by SEM and staining methods with Nile red and Nile blue, respectively; strains were also analysed by the fluorescence imaging technique. Among them, JCCOL25.8 strain showed the highest production of PHA's reason why phenotypic and genotypic characterization was performed; this strain was identified as Halomonas nitroreducens. Polymer produced by this strain was characterized by FTIR, DSC, GPC and EDX spectroscopy. Results indicated that the biosynthesized polymer was polyhydroxybutyrate (PHB) which had a melting peak at 170°C and a crystallinity percentage of about 36%. Based on phenotypic and genotypic aspects, JCCOL25.8 strain was identified as H. nitroreducens and it was capable to accumulate PHB. To our knowledge, there is only one study published on the biosynthesis of PHA's by H. nitroreducens strains, although the characterization of the obtained polymer was not reported.

An experimental point of view on hydration/solvation in halophilic proteins

Protein-solvent interactions govern the behaviors of proteins isolated from extreme halophiles. In this work, we compared the solvent envelopes of two orthologous tetrameric malate dehydrogenases (MalDHs) from halophilic and non-halophilic bacteria. The crystal structure of the MalDH from the non-halophilic bacterium Chloroflexus aurantiacus (Ca MalDH) solved, de novo, at 1.7 Å resolution exhibits numerous water molecules in its solvation shell. We observed that a large number of these water molecules are arranged in pentagonal polygons in the first hydration shell of Ca MalDH. Some of them are clustered in large networks, which cover non-polar amino acid surface. The crystal structure of MalDH from the extreme halophilic bacterium Salinibacter ruber (Sr) solved at 1.55 Å resolution shows that its surface is strongly enriched in acidic amino acids. The structural comparison of these two models is the first direct observation of the relative impact of acidic surface enrichment on the water structure organization between a halophilic protein and its non-adapted counterpart. The data show that surface acidic amino acids disrupt pentagonal water networks in the hydration shell. These crystallographic observations are discussed with respect to halophilic protein behaviors in solution

Synthesis of bacteriorhodopsin by Halobacterium sodomense K91r

The light-dependent protein bacteriorhodopsin deserves special attention among physiologically active compounds from halophilic Archeans. It is associated with the development of a new type of information-storage system, the creation of an artificial eye, and the use as biologically active additives with clearly pronounced antioxidant and radioprotector properties [1]. We isolated previously two strains of extreme halophilic bacteria from Barsakelmes salt marsh (South Sub-Aral) that were capable of synthesizing carotinoid pigments [2]. The goal of the present study was to identify and determine quantitatively bacteriorhodopsin in cells of the previously isolated strain Halobacterium sodomense K91r and to study the protein accumulation as a function of culture age. The capability of cells of an extreme halophile to synthesize bacteriorhodopsin was studied for the first time in Uzbekistan. Cell-free extract of H. sodomense K91r was obtained by the standard method using CFE buffer [3]. The composition of the resulting protein extract was analyzed by electrophoresis under denaturing conditions according to Laemmli [4]. The electrophoregram of the studied extract showed 16 bands corresponding to proteins of molecular weights in the range 110–7 kDa. Among them, a protein band of molecular weight of the order of 26 kDa was observed. This corresponded to bacteriorhodopsin standard (Fluka). The identification of this protein in the extract from the halophilic strain was promising for further work to isolate and purify bacteriorhodopsin. The quantitative content of the synthesized bacteriorhodopsin was determined as a function of the cultivation time of the strain and the composition of the growth medium for No. 44 and Braun liquid media using spectrophotometry based on the light-sensitivity of the protein [5]. For this, cells were collected from growth medium (2 mL) by centrifugation and disintegrated by distilled water containing DNAase 1 (0.01 mg). The lysate was stirred until homogeneous and treated with NaOH (4 M) and NH4OH (4 M) in a 9:0.5:0.5 (lysate:NaOH:NH4OH) ratio in the dark. Absorption at 568 nm (A568) was first measured in the dark (A0 568). The mixture was exposed to light for 24 h. The absorption was measured again (A 24 568). Considering that the molecular weight of bacteriorhodopsin is 26 kDa and the molar extinction coefficient is 63,000 M–1·cm–1, the bacteriorhodopsin concentration was determined from the equation BR (g/L) = 26,000 (A0 568 – A 24 568)/63,000. Bacteriorhodopsin began to accumulate heavily starting on the 5–6th day after the start of cultivation. This was due to an increase of the NaCl concentration in the medium and; therefore, a decrease of the aeration of the growth medium and a switch to the conversion of light energy into chemical mediated by bacteriorhodopsin. Despite the fact that H. sodomense K91r biomass grew more vigorously in Braun medium, this did not affect the accumulation of bacteriorhodopsin. A study of bacteriorhodopsin accumulation during growth of H. sodomense K91r found that the yield of pigment was greater during cultivation in No. 44 medium than in Braun medium. The values were 11.3 and 10 mg/L, respectively, on the 11th and 10th day of cultivation. The bacteriorhodopsin concentration decreased sharply on the 14th day of cultivation. Bacteriorhodopsin accumulation by the local strain of H. sodomense K91r did not depend directly on the amount of biomass and the medium composition but moreso on several other factors such as the illumination and age of the cultivation medium. Thus, our data enabled the cultivation conditions of the microorganism to be optimized in order to increase the synthesis of the target protein.

Culture alternative medium for the growth of extreme halophilic bacteria in fish products

Halotolerant or halophilic (Archaeabacteria) microorganisms can be found in salted and ripening fish products that are not affected by salt. They can be moderate or extremely halophilic bacteria. The extremely halophilic bacteria require between 15-30% of NaCl for growth. The extremely halophilic archaeobacteria may be selectively isolated in different media. The aim of this work was to determine the effectiveness of the Salt-Agar-Milk medium, a medium modified in our laboratory through the addition of MgSO4 and KCl - named SAMm, and its effect on the bacterial growth by means of comparison with other media, with and without milk, determining time of incubation and counting. Two samples of salted fish from local fish salting factories and two laboratory strains were used. The factory samples were matured anchovy and anchovy fillets in oil, and the laboratory strains were: Haloarcula spp. (proteolytic) and Halococcus spp. (non-proteolytic). The following media were alternatively used for the isolation of extremely halophilic bacteria: IRAM; Formulation of Gibbons and collaborators, Cod Milk agar, and SAMm. IRAM and Gibbons were also used enriched with milk. In the SAMm medium, there were obtained count values similar or higher than the ones of the traditional media; besides the simplicity of its elaboration, the possibility to obtain positive results two or three days earlier also added to its benefit. Consequently, it can be considered an alternative to the media traditionally used for the studied halophilic bacteria.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber ( Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K m for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.

Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP).

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community "genome" had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

Bacteriorhodopsin: the mechanism of 2D-array formation and the structure of retinal in the protein

Bacteriorhodopsin, the light driven proton pump of the extreme halophilic bacterium H. salinarium, is an integral membrane protein ( M r ca. 26000) which forms 2D arrays in the purple membrane of the bacterium. It is this feature which has permitted the use of electron diffraction methods to resolve the protein structure to some degree of atomic detail, although the prosthetic group has not been fully resolved. However, the features which induce the protein to form these arrays have not been previously clarified. We have now shown that the protein array formation is driven by specific interaction of the protein with the charged phospholipid, phosphatidyl glycerol phosphate (or the sulphate derivative), a major (ca. 60%) lipid of the bacterial host membrane. In addition, in an effort to provide further structural information about the chromophore, retinal, of this protein, the orientation of the individual methyl groups of retinal have been determined from solid state deuterium NMR studies of the deuterated chromophore when in the protein binding site. This approach to structural resolution of the prosthetic group is ab initio, agrees with other studies on the chromophore and resolves new features of the bound retinal to a high degree (±2°) of precision. Here, these two studies on this integral membrane protein will be reviewed.

Life in Unusual Environments: Progress in Understanding the Structure and Function of Enzymes from Extreme Halophilic Bacteria

Extreme halophilic archaea are saturated with salt and the intracellular electrolyte concentration exceeds that of the extracellular environment. Enzymes and other proteins from extreme halophilic archaea have been purified for many years and studied by biochemical and biophysical solution methodologies. They are active and stable at multimolar salt concentrations and denature below 2 to 3 M NaCl or KCl, Adaptation to these high concentrations of salt, genetic and evolutionary aspects, and the possibility of biotechnological applications are problems of considerable interest, Since the status of this fascinating field of research was reviewed in 1992, malate dehydrogenase from Haloarcula marismortui, now known to be a tetramer, was sequenced, its gene was cloned and expressed in active form, and its physical properties were redefined, A single mutation of Arg100 (in the enzyme active site) to Gin switched the enzyme specificity from malate to lactate dehydrogenase, Recent determination of its molecular structure by X-ray crystallography (O, Dym et al., in press) provides an exciting basis for the understanding of the structure and function of extreme halophilic enzymes, A major problem which so far has not been tackled in the study of extreme halophilic archaea is the understanding of protein nucleic acid interactions which are essential for the performance of biological function, Whereas the stability and activity of enzymes and other proteins can be modified to perform at high salt concentrations by use of currently known structural concepts, the existence of meaningful protein nucleic acid interactions in physiological concentrations of 4 to 5 M KCI constitutes an unsolved enigma worth intensive investigation.