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At least 19 recordsLinked to original sources

PubMed2003

Construction and usage of a onefold-coverage shotgun DNA microarray to characterize the metabolism of the archaeon Haloferax volcanii.

Haloferax volcanii is a moderately halophilic archaeon that can grow aerobically and anaerobically with a variety of substrates. We undertook a novel approach for the characterization of metabolic adaptations, i.e. transcriptome analysis with a onefold-coverage shotgun DNA microarray. A genomic library was constructed and converted into a polymerase chain reaction (PCR) product library, which was used to print two DNA microarrays, a 960-spot test array used for optimization of microarray analysis and a 2880-spot onefold-coverage array. H. volcanii cultures were shifted from casamino acid-based metabolism to glucose-based metabolism, and the transcriptome changes were analysed with the onefold-coverage array at five time points covering the transition phase and the onset of exponential growth with the new carbon source. About 10% of all genes were found to be more than 2.5-fold regulated at at least one time point. The genes fall into five clusters of kinetically co-regulated genes. For members of all five clusters, the results were verified by Northern blot analyses. The identity of the regulated genes was determined by sequencing. Many co-regulated genes encode proteins of common functions. Expected as well as a variety of unexpected findings allowed predictions about the central metabolism, the transport capacity and the cellular composition of H. volcanii growing on casamino acids and on glucose. The microarray analyses are in accordance with the growth rates and ribosome contents of H. volcanii growing on the two carbon sources. Analysis of the results revealed that onefold-coverage shotgun DNA microarrays are well suited to characterize the regulation of metabolic pathways as well as protein complexes in response to changes in environmental conditions.

Gene Expression Profiling↗

PubMed2005

Complete reversal of coenzyme specificity of isocitrate dehydrogenase from Haloferax volcanii.

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP+ to NAD+ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.

Amino Acid Sequence↗

PubMed2003

Subunit topology of two 20S proteasomes from Haloferax volcanii.

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (alpha1, alpha2, and beta) which are classified in the 20S proteasome superfamily. The alpha1 and beta proteins alone form active 20S proteasomes; the role of alpha2, however, is not clear. To address this, alpha2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The alpha2 protein copurified with alpha1 and beta in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described alpha1-beta proteasome. Supplementing buffers with 10 mM CaCl(2) stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the alpha1-beta and alpha1-alpha2-beta proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the alpha1-beta proteasome was a symmetrical cylinder with alpha1 rings on each end. In contrast, the alpha1-alpha2-beta proteasome appeared to be asymmetrical with homo-oligomeric alpha1 and alpha2 rings positioned on separate ends. Inter-alpha-subunit contacts were only detected when the ratio of alpha1 to alpha2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of alpha proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.

Blotting, Western↗

PubMed1997

Osmotically induced response in representatives of halophilic prokaryotes: the bacterium Halomonas elongata and the archaeon Haloferax volcanii.

Haloferax volcanii and Halomonas elongata have been selected as representatives of halophilic Archaea and Bacteria, respectively, to analyze the responses to various osmolarities at the protein synthesis level. We have identified a set of high-salt-related proteins (39, 24, 20, and 15.5 kDa in H. elongata; 70, 68, 48, and 16 kDa in H. volcanii) whose synthesis rates increased with increasing salinities. A different set of proteins (60, 42, 15, and 6 kDa for H. elongata; 63, 44, 34, 18, 17, and 6 kDa for H. volcanii), some unique for low salinities, was induced under low-salt conditions. For both organisms, and especially for the haloarchaeon, adaptation to low-salt conditions involved a stronger and more specific response than adaptation to high-salt conditions, indicating that unique mechanisms may have evolved for low-salinity adaptation. In the case of H. volcanii, proteins with a typical transient response to osmotic shock, induced by both hypo- and hyperosmotic conditions, probably corresponding to described heat shock proteins and showing the characteristics of general stress proteins, have also been identified. Cell recovery after a shift to low salinities was immediate in both organisms. In contrast, adaptation to higher salinities in both cases involved a lag period during which growth and general protein synthesis were halted, although the high-salt-related proteins were induced rapidly. In H. volcanii, this lag period corresponded exactly to the time needed for cells to accumulate adequate intracellular potassium concentrations, while extrusion of potassium after the down-shift was immediate. Thus, reaching osmotic balance must be the main limiting factor for recovery of cell functions after the variation in salinity.

Adaptation, Physiological↗

PubMed1995

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↗

PubMed2002

Functional role for a 2-oxo acid dehydrogenase in the halophilic archaeon Haloferax volcanii.

The archaeon Haloferax volcanii was previously shown to contain and transcribe the genes for a 2-oxo acid dehydrogenase (OADH) complex, but their presence remained a mystery because no enzymatic activity with any of the known OADH substrates could be found, and an inactivation of one of the genes did not lead to any phenotype. Here we report the identification of an additional oadh gene cluster in the genome of H. volcanii. In contrast to previously known oadh loci, it contains three genes, oadh2A1, oadh2A2, and oadh2ld, with coding capacity for the E1alpha and E1beta subunits and an unattached lipoyl domain, but it is devoid of the genes for a complete E2 and an E3. The genes were isolated by complementation of a nitrate respiration-deficient mutant of H. volcanii and therefore were shown to be functional in vivo. Phylogenetic analyses revealed that the deduced E1alpha and E1beta subunits of OADH2 group with bacterial acetoin dehydrogenases but not with the OADH1 subunits, and thus, H. volcanii has obtained the two gene groups independently. Comparison of the wild type and the mutant allowed us to exclude a function of OADH2 in the aerobic or anaerobic degradation of acetoin or glucose. Instead, it could be shown that OADH2 is important during nitrate-respirative growth on Casamino Acids. Many physiological and biochemical experiments failed to indicate that OADH2 uses any of the previously known OADH substrates. Growth potentials of the mutant were markedly different in media with a single carbon source versus media with mixed carbon sources.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2002

Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C. Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions.

Adenosine Triphosphatases↗

PubMed2004

Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii produces three different proteins (alpha1, alpha2, and beta) that assemble into at least two 20S proteasome isoforms. This work reports the cloning and sequencing of two H. volcanii proteasome-activating nucleotidase (PAN) genes (panA and panB). The deduced PAN proteins were 60% identical with Walker A and B motifs and a second region of homology typical of AAA ATPases. The most significant region of divergence was the N terminus predicted to adopt a coiled-coil conformation involved in substrate recognition. Of the five proteasomal proteins, the alpha1, beta, and PanA proteins were the most abundant. Differential regulation of all five genes was observed, with a four- to eightfold increase in mRNA levels as cells entered stationary phase. In parallel with this mRNA increase, the protein levels of PanB and alpha2 increased severalfold during the transition from exponential growth to stationary phase, suggesting that these protein levels are regulated at least in part by mechanisms that control transcript levels. In contrast, the beta and PanA protein levels remained relatively constant, while the alpha1 protein levels exhibited only a modest increase. This lack of correlation between the mRNA and protein levels for alpha1, beta, and PanA suggests posttranscriptional mechanisms are involved in regulating the levels of these major proteasomal proteins. Together these results support a model in which the cell regulates the ratio of the different 20S proteasome and PAN proteins to modulate the structure and ultimately the function of this central energy-dependent proteolytic system.

Adenosine Triphosphatases↗

PubMed2001

Post-translational modification of the S-layer glycoprotein occurs following translocation across the plasma membrane of the haloarchaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii is surrounded by a protein shell solely comprised of the S-layer glycoprotein. While the gene sequence and glycosylation pattern of the protein and indeed the three-dimensional structure of the surface layer formed by the protein have been described, little is known of the biosynthesis of the S-layer glycoprotein. In the following, pulse-chase radiolabeling and cell-fractionation studies were employed to reveal that newly synthesized S-layer glycoprotein undergoes a maturation step following translocation of the protein across the plasma membrane. The processing step, detected as an increase in the apparent molecular mass of the S-layer glycoprotein, is unaffected by inhibition of protein synthesis and is apparently unrelated to glycosylation of the protein. Maturation requires the presence of magnesium ions, involved in membrane association of the S-layer glycoprotein, and results in increased hydrophobicity of the protein as revealed by enhanced detergent binding. Thus, along with protein glycosylation, additional post-translational modifications apparently occur on the external face of the haloarchaeal plasma membrane, the proposed topological homologue of the lumenal face of the eukaryal endoplasmic reticulum membrane.

Amino Acid Sequence↗

PubMedDate not supplied

Isolation, characterization, and substrate specificity of the plasma membrane ATPase of the hlophilic achaeon Haloferax volcanii.

Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg2+ or Mn2+, high concentrations of NaCl, a pH value of 9, and high temperatures with an optimum at 60 degrees C. We have not found any phosphatase activity in the preparations. We developed a purification method for the isolated enzyme with an enrichment factor of 90. SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa. N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cyteines, inhibited the enzyme slightly. Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides. Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the A1 part of the halophilic ATPase. The described genes are organized in an operon in the order D, C, E, B, A, named alphabetically according to their decreasing size. The deduced products of 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification of the ATPase. Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn2+ higher rates of ATP hydrolysis could be observed than in presence of Mg2+, but free manganese ions inhibited the enzyme activity of the ATPase. Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of Michaelis-Menten constants for ATP in the hydrolysis direction of 1 mM in presence of MgCl2 and 0.24 mM in presence of MnCl2. Sodium chloride concentrations in the molar range induce changes in KM by a factor of about 10. The enzyme is specific for ATP; other nucleotides including GTP and ADP are competitive inhibitors of ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed2006

HMG-CoA reductase is regulated by salinity at the level of transcription in Haloferax volcanii.

The moderately halophilic archaeon Haloferax volcanii was surveyed for protein profile changes correlated with growth at high and low salinity. A single polypeptide with an approximate mass of 46 kDa was conspicuously more abundant during growth at high salinity. This protein was identified as HMG-CoA reductase (HMGR), encoded by the hmgR gene. HMGR is a key enzyme in the mevalonate pathway of isoprenoid biosynthesis, the sole route in haloarchaea for lipid and carotenoid production. Enzymatic assays confirmed that HMGR activity is more abundant in cells grown at high salinity. Low salt cultures of H. volcanii contained lower amounts of hmgR transcript compared to cells grown in high salt suggesting that the observed regulation occurs at the level of transcription. Paradoxically, both lipid and carotenoid content decreased in H. volcanii grown at high salinity despite the increased levels of HMGR specific activity. To our knowledge, this is the first report demonstrating that the expression of HMGR is regulated in response to non-optimal salinity in a halophilic archaeon.

Adaptation, Physiological↗

PubMed2000

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases.

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.

Amino Acid Sequence↗

PubMed1990

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).

Amino Acid Sequence↗

PubMed1995

A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA.

Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively. At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop. Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii. Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process. The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase. This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala). In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37. Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.

Adenosine↗

PubMed2002

Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii.

By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE.

Archaeal Proteins↗

PubMed1992

Transcriptionally active regions in the genome of the archaebacterium Haloferax volcanii.

Transcriptionally active regions of the Haloferax volcanii genome were mapped by hybridization of radiolabeled cDNA to Southern blots of our minimal set of overlapping cosmid clones covering 96% of the 4.1-Mbp genome. Transcription during exponential growth occurred in nearly every region of the 2,920-kbp chromosome. Large parts of the 690- and the 86-kbp plasmids were transcribed, but the 440-kbp plasmid showed little expression. Transcription after a 40-min heat shock at 65 degrees C was generally reduced, apart from a small set of strongly expressed loci all situated on the chromosome.

Archaea↗

PubMed1995

Nucleotide sequence of the ATPase A- and B-subunits of the halophilic archaebacterium Haloferax volcanii and characterization of the enzyme.

Using PCR with degenerate oligonucleotides, we amplified two conserved regions of the plasma membrane ATPase A (alpha)- and B (beta)-subunit-encoding genes from Haloferax volcanii, a halophilic archaeon. The amplified fragments were cloned and sequenced, and then used as homologous probes to clone genomic restriction fragments, one of which contained the nearly complete atpA- and atpB-gene. To complete the latter one, we sequenced a region of an overlapping fragment using synthetic oligonucleotides as primers. The deduced amino-acid sequence showed a high degree of similarity with the A and B sequences of Halobacterium halobium-ATPase, and a relatively high degree with Daucus carota V-ATPase, but less similarity to F-ATPase alpha- and beta-subunits. Like in V-ATPases, the A-subunit is more related to the catalytic F-ATPase beta-subunit, whereas B corresponds to alpha. Cross-reaction of antibodies against CF1-alpha (synthetic peptide) with B and CF1-beta with A of Haloferax volcanii confirmed this finding. The ATPase of Haloferax volcanii is a halophilic enzyme, the amino-acid sequences contain about 20% negatively charged residues. This is discussed in terms of adaption to hypersaline conditions. The enzyme is specific for ATP, we determined KM values for ATP in presence of Mn2+ and Mg2+, respectively. Other nucleotides, including GTP and ADP are competitive inhibitors of ATP hydrolysis. Despite of the high degree of similarity to the Halobacterium enzyme, the ATPase showed no sensitivity to most of the tested inhibitors of ATP hydrolysis. NEM, a specific inhibitor for V-ATPases inhibited the enzyme only slightly. Bafilomycin, NBD-Cl and nitrate showed no effect. These results will be discussed in context with some specific differences in the primary structure of the Haloferax A-subunit.

Amino Acid Sequence↗

PubMed2002

Cytochrome aa(3) in Haloferax volcanii.

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.

Archaeal Proteins↗