Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.     

Halophile aldehyde dehydrogenase from Halobacterium salinarum

Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.     

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.     

The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.     

Functional expression of green fluorescent protein derivatives in Halobacterium salinarum

We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.     

A two-alpha-helix extra domain mediates the halophilic character of a plant-type ferredoxin from halophilic archaea

The [2Fe-2S] ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibits a high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 amino acids near its N-terminus which is extremely rich in acidic amino acids. Unfolding studies reveal that HsFdx has an unfolding temperature of approximately 85 degrees C in 4.3 M NaCl, but of only 50 degrees C in low salinity, revealing its halophilic character. The three-dimensional structure of HsFdx was determined by NMR spectroscopy, resulting in a backbone rmsd of 0.6 A for the diamagnetic regions of the protein. Whereas the overall structure of HsFdx is very similar to that of the plant-type ferredoxins, two additional alpha-helices are found in the acidic extra domain. (15)N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility of residues is similar throughout the molecule. Monitoring protein denaturation by NMR did not reveal differences between the core fold and the acidic domain, suggesting a cooperative unfolding of both parts of the molecule. A mutant of the HsFdx in which the acidic domain is replaced with a short loop of the nonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern. The halophilic wild-type protein is readily expressed in large amounts in H. salinarum, but not in Escherichia coli, whereas the mutant ferredoxin could only be overexpressed in E. coli. The salt concentration was also found to play a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the cluster in the mutant protein proceeds efficiently in low salt. These findings suggest that the acidic domain mediates the halophilic character which is reflected in its thermostability, the exclusive expression in H. salinarum, and the ability to efficiently reconstitute the iron-sulfur cluster only at high salt concentrations.     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.     

Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer

A Co(2+)-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been developed. The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT-Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively. The His-tagged HemAT-Hs can be purified rapidly using either low or high ionic strength buffers. Purified His-tagged HemAT-Hs in high or low salt buffers demonstrated no difference in spectral characteristics and retained reversible oxygen binding capacity. This fast-flow Co(2+)-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophysical and structural studies.     

Increased stability of malate dehydrogenase from Halobacterium salinarum at low salt concentration in reverse micelles

The stability of malate dehydrogenase (hMDH) from Halobacterium salinarum in aqueous medium at low salt concentrations (1 and 0.5 M NaCl) was studied at 4 degrees and 25 degrees C. The results showed that hMDH was more stable at the higher salt concentration and the low temperature. hMDH was introduced into reverse micelles of hexadecyltrimethylammonium bromide in cyclohexane with 1-butanol as co-surfactant. The hMDH stability in this system was studied at two omega(0) ([H(2)O]/[surfactant]) values and the effects of salt concentration, presence of substrate and dilution before or after its introduction into reverse micelles were examined. The results showed that the half-life of hMDH dissolved in buffer with 1 M NaCl was 12-50 days in reverse micelles (depending on the various conditions), in contrast to only about 1 day in aqueous medium at 25 degrees C. These observations indicate that reverse micelles provide a microenvironment that allows a much greater stability of this enzyme compared with an aqueous medium.     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

Tolerance of Mesorhizobium type strains to different environmental stresses

The symbiosis between rhizobia and legumes is affected by different environmental conditions. Our aims were to evaluate stress tolerance of Mesorhizobium species and investigate species-specific stress response mechanisms. Tolerance of Mesorhizobium type strains to temperature, salt and pH stress was evaluated. Mesorhizobium thiogangeticum showed highest growth with 1.5% NaCl and Mesorhizobium ciceri at pH 5. Mesorhizobium plurifarium showed higher growth at 37°C. SDS-PAGE analysis revealed changes in the protein profiles, namely the overexpression of a 60 kDa protein, following heat stress. Under salt stress, five overexpressed proteins were identified in M. plurifarium and M. thiogangeticum. Northern analysis revealed an increase in groEL expression in Mesorhizobium huakuii and Mesorhizobium septentrionale after heat shock; by contrast, a decrease was detected in Mesorhizobium albiziae and M. thiogangeticum, upon salt shock. A high diversity in tolerance to temperature, salt and pH stress was detected among Mesorhizobium species. M. thiogangeticum and M. ciceri are moderately halophilic and acidophilic, respectively. Several proteins, overproduced in different strains, may be involved in stress tolerance. groEL expression increased upon heat and decreased upon salt shock. To our knowledge, this is the first study focusing tolerance to temperature, salt and pH stress, as well as groEL expression, in Mesorhizobium type strains.     

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.     

Protease Formation by a Moderately Halophilic Bacillus Strain

A moderately halophilic strain of Bacillus, isolated from unrefined solar salt, was capable of growth in the presence of 4 M NaCl. Maximal growth was obtained in a medium containing 1 to 2 M NaCl. The organism produced protease when cultivated aerobically in media containing 0 to 3 M NaCl or 0 to 2 M KCl. The protease activity was optimal at 0.5 M NaCl and 0.75 M KCl.     

Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.