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PubMed1993

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.

Cell Membrane↗
PubMed1993

A C-terminal truncation results in high-level expression of the functional photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium.

Expression of the gene encoding the halobacterial photoreceptor sensory rhodopsin I (SRI), sopI, was studied by means of homologous gene targeting. A sopI- Halobacterium salinarium mutant strain was constructed by homologous replacement of sopI with a novobiocin-resistant gyrB from Haloferax Aa 2.2. Cells bearing gyrB were resistant to novobiocin, indicating that the Haloferax gene is functional in H. salinarium. Complementation of this deletion strain with sopI fused to the bacterio-opsin promoter resulted in the recovery of all phenotypical attributes of SRI. This establishes the first direct correlation between sopI and the function of its gene product. In the complemented deletion strain, functional expression of sopI occurred from the bop locus, where sopI had integrated by homologous recombination. This shows that cotranscription of sopI and the gene encoding the SRI signal transducer, htrI, which is found in the wild type, is not a prerequisite for photosensory activity. Deletion of the last 43 bp at the 3' end of sopI resulted in a 10-fold increase in the amount of SRI, without affecting the activity of the pigment. The mRNA level of the truncated gene was not affected as compared to that of the wild type. We propose that regulation occurs at the protein level, probably through a negative determinant of protein stability located in the C-terminus of SRI. Replacement of the last 28 amino acids of bacteriorhodopsin by the last 29 amino acids of SRI results in a decrease of the bacteriorhodopsin, supporting our observations. The C-terminus of SRI is the first domain with a downregulating influence on protein levels thus far identified in H. salinarium. The system for SRI overexpression we present here greatly facilitates biochemical and biophysical studies on the photoreceptor and allows investigation of the molecular interactions underlying the signal transduction chain of halobacterial phototaxis.

Amino Acid Sequence↗
PubMed1995

Effects of upstream deletions on light- and oxygen-regulated bacterio-opsin gene expression in Halobacterium halobium.

The bacterio-opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light- and oxygen-regulated bop gene expression was determined to be 54 bp.

Bacteriorhodopsins↗
PubMed1995

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes.

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF-M. The 5'-terminus of the p-gvpF-M mRNA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac- species Haloferax volcanii as recipient. Construct delta A containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac- transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac++). Similarly, double transformants containing p-gvpD-M plus p-gvpACNO, or p-gvpG-M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED-ACNO were Vac++. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac-, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF-M plus p-gvpACNO (delta DE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF-M mRNA could be suggested for p-gvpD and the 5'-region of its mRNA.

Archaeal Proteins↗
PubMed2005

MpcT is the transducer for membrane potential changes in Halobacterium salinarum.

In Halobacterium salinarum mutants containing either of the light-driven ion pumps bacteriorhodopsin (H(+)) or halorhodopsin (Cl(-)) as their only retinal protein, a decrease of irradiance in the absence of respiration causes a phototactic response. The conversion of the causal event, a decrease of proton motive force across the cell membrane, into a reversal of flagellar motor rotational direction was expected to involve a transducer. Via deletion analysis of all 18 known and putative halobacterial transducer (htr) genes, we found that Htr14, a methylatable membrane-bound transducer lacking an extracellular domain, mediates the biological response, which includes adaptive methylation. Based on a minimal stimulus length of 200 ms and the determined cytoplasmic buffering capacity, we conclude that the change in the membrane potential (DeltaPsi), and not that of the internal pH, is the signal-generating event. Htr14 was therefore renamed to Membrane potential change Transducer, or MpcT. It is the first transducer for which the causative stimulus could be narrowed to a change in DeltaPsi, as opposed to a change in pH or cellular redox state.

Adaptation, Physiological↗
PubMed2006

Translation initiation with GUC codon in the archaeon Halobacterium salinarum: implications for translation of leaderless mRNA and strict correlation between translation initiation and presence of mRNA.

We have investigated whether anticodon sequence mutant of an archaeal initiator tRNA can initiate protein synthesis using reporter genes carrying mutations in the initiation codon. Halobacterium salinarum was used as the model organism and the bacterio-opsin gene (bop), which encodes the precursor of the protein component of the purple membrane protein bacterio-opsin (Bop), was chosen as the reporter. We demonstrate that a CAU to GAC anticodon sequence mutant of Haloferax volcanii initiator tRNA can initiate Bop protein synthesis using GUC as the initiation codon in H. salinarum. We generated four mutant bop genes, each carrying the AUG to GUC initiation codon mutation, with or without a compensatory mutation to maintain a predicted stem-loop structure at the 5'-end of the bop mRNA, and with or without mutations to test translation initiation at a site corresponding to the amino terminus of mature bacterio-opsin. H. salinarum chromosomal recombinants containing these mutant genes were phenotypically Pum- (purple membrane negative). Upon transformation with a plasmid carrying the mutant initiator tRNA gene, only strains designed to maintain the bop mRNA stem-loop structure produced Bop and were phenotypically Pum+ as indicated by purple colony colour, and immunoblotting and spectral analysis of cell extracts. Thus GUC can serve as an initiation codon in archaea and the stem-loop structure in the bop mRNA is important for translation. Interestingly, for the same mutant mRNA, only transformants that produce Bop protein contain bop mRNA. These results suggest either a strong coupling between translation and mRNA stability or strong transcriptional polarity in H. salinarum.

5' Untranslated Regions↗
PubMed1978

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.

Adenosine Triphosphatases↗
PubMed1978

Potassium uniport and ATP synthesis in Halobacterium halobium.

Light-driven potassium ion uptake in Halobacterium halobium is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy.

Adenosine Diphosphate↗
PubMed1978

DNA-dependent RNA polymerase from Halobacterium halobium.

DNA-dependent RNA polymerase core enzyme was isolated from Halobacterium halobium. The purification is based on the finding that the enzyme is stable in 40% (v/v) glycerol, in the presence of 0.05 M MgCl2 and involves adsorption of contaminants to DEAE-cellulose, precipitation of the complex of polymerase with DNA by streptomycin sulfate, chromatography over Biogel and affinity chromatography over heparin-Sepharose or heparin-cellulose. The enzyme consists of four or five different subunits. The composition formula was estimated as (150000) (86000)2 (72000)2 (49000)3 or 2; there may be one or two different 49000-Mr subunits. RNA synthesis requires a template. Denatured DNA is more efficient than native DNA. The transcription of native DNA is specifically stimulated by the addition of a possibly sigma-like factor eluted from DEAE-cellulose. The fidelity of transcription is indicated by the absolute requirement for UTP besides ATP with poly[d(A-T)] as the template.

DNA-Directed RNA Polymerases↗
PubMed1980

Preparation and characterization of inverted cell envelopes of Halobacterium halobium.

Inside-out cell envelopes from Halobacterium halobium R1M1 have been isolated by sonication and subsequent sucrose-density-gradient fractionation. The inverted cell envelopes transport protons from the exterior of the vesicles by a light-dependent, uncoupler-inhibited process. Enzymes usually facing the inside of the cell are exposed to the external medium in this preparation.

Bacteriorhodopsins↗
PubMed1981

Two elongation factors from the extremely halophilic archaebacterium Halobacterium cutirubrum. Assay systems and purification at high salt concentrations.

Two peptide chain elongation factors from Halobacterium cutirubrum were purified nearly to homogeneity. They were identified and characterized by three assay systems working at very high salt concentrations: poly(Phe) synthesis, GDP binding and ADP-ribosylation. The purification procedure consisted of Sepharose 4B chromatography with a decreasing ammonium sulfate gradient, gel filtration on Sephadex G-100 in the presence of (NH4)2SO4 and, for each of the two separated factors, an independent adsorption chromatography on hydroxyapatite. The factors were absolutely dependent on high salt concentrations for stability and activity. Both factors (I and II) complement each other to give fully active poly(Phe) synthesis, which is totally inhibited by puromycin and anisomycin. Factor I (Mr 51 000) is a major protein of the cell-free extract. It binds GDP, which can be displaced by GTP only to a small extent. The function of factor I in poly(Phe) synthesis is not impaired by high concentrations of kirromycin. The observed characteristics resemble partly those of prokaryotic EF-Tu and partly those of eukaryotic EF-1. Factor II (Mr 111 000) can be ADP-ribosylated by diphtheria toxin, and thus was identified as an EF-2-type elongation factor.

Guanosine Diphosphate↗
PubMed1981

Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium.

Pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase were obtained from cell-free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting-out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of non-identical subunits of Mr 86000 and 42000 in the case of the pyruvate-degrading enzyme and of 88000 and 36000 in the case of the 20 -oxogluatarate-degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe-4S]2 + (2 + , 1+) clusters and two molecules of thiamin diphosphate. Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2-oxoacid:ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2-oxoacid dehydrogenase multienzyme complexes which commonly occur in anaerobic organisms.

Amino Acids↗
PubMed1981

The catalytic mechanism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. One-electron transfer at two distinct steps of the catalytic cycle.

The catalytic cycle of the 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium was investigated. The first step is binding of the 2-oxoacid to the enzyme followed by decarboxylation and transfer of one electron to the [4Fe-4S] cluster of the functional unit. The cluster is then reoxidized by ferredoxin or, in the absence of the physiological electron acceptor, by oxygen. In the resulting stable enzyme-intermediate radical the decarboxylation product of the 2-oxoacid remains tightly bound until reaction with coenzyme A caused formation of acyl-CoA and concomitant transfer of the second electron to the cluster, which again is reoxidized by ferredoxin or oxygen. After purification, part of the enzyme molecules still contain the intermediate radical. Enzyme preparations either free of radical or containing enhanced amounts are obtained by treatment with coenzyme A or 2-oxoacid, respectively. Whenever the radical is present in an enzyme molecule the respective binding site for the 2-oxoacid is blocked.

Binding Sites↗
PubMed1983

A form of the DNA-dependent RNA polymerase of Halobacterium halobium, containing an additional component, is able to transcribe native DNA.

A revised procedure for the purification of DNA-dependent RNA polymerase from Halobacterium halobium, including two-phase partition, yields pure, highly active and absolutely DNA-dependent enzyme. Two forms of the enzyme, one containing, the other not containing a previously not observed component, epsilon, show striking differences in activity. RNA polymerase without component epsilon has a significant activity on poly[d(A-T)] but only insignificant activity on all other templates. The enzyme containing a stoichiometric amount of component epsilon transcribes poly[d(A-T)] and native templates efficiently.

Chemical Phenomena↗
PubMed1985

An alpha-like DNA polymerase from Halobacterium halobium.

Two DNA polymerases have been isolated from extracts of Halobacterium halobium, one having a sedimentation coefficient of 11 S, designated as alpha-like polymerase and possessing the following characteristics. It is sensitive to both aphidicolin and N-ethylmaleimide but indifferent to the presence of a dideoxynucleoside triphosphate. Therefore this polymerase is very similar to the alpha DNA polymerase of eukaryotes. The enzyme requires 5 M NaCl for maximum activity. The other polymerase has a sedimentation coefficient of 4.4 S and is resistant to both aphidicolin and N-ethylmaleimide. However, it is inhibited by a dideoxynucleoside triphosphate.

Aphidicolin↗
PubMed1988

Purification and characterization of peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium.

A procedure is described for the purification of the archaebacterial peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium. The enrichment was about 530-fold, the obtained preparation practically homogeneous as judged by SDS-PAGE. The poly(U)-dependent poly(Phe) synthesis was completely dependent on aEF-2 in the presence of partially purified aEF-1, and the activity was equivalent to a poly(Phe)-synthesizing system containing unfractionated S-100 enzymes. aEF-2 consists of a single peptide with a relative molecular mass of 125,000 +/- 3000 and 100,000 +/- 3000 as determined by SDS-PAGE and gel filtration on Sephadex G-200 respectively. The isoelectric point was 5.7. The amino acid composition analysis indicated the predominance of acidic amino acids (aspartic acid and glutamic acid) and the low content of hydrophobic amino acid (phenylalanine) as compared with those of eukaryotes and prokaryotes. The factor was stable in a pH range from 6 to 8. 2-Mercaptoethanol and GTP but not GDP markedly protected aEF-2 from heat denaturation at 52 degrees C. aEF-2 became inactivated and insensitive to ADP-ribosylation by diphtheria toxin at low ionic strength but could be renatured by increasing ionic strength. Obviously higher concentrations of salts contribute to the conformational stability of aEF-2.

Adenosine Diphosphate Ribose↗
PubMed1988

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

Amino Acid Sequence↗
PubMed1988

DNA primase activity found in an alpha-like DNA polymerase obtained from Halobacterium halobium.

An aphidicolin-sensitive DNA polymerase was purified from extracts of Halobacterium halobium. The analysis of this alpha-like DNA polymerase on polyacrylamide gels under denaturing conditions revealed two peptides with molecular masses of 70 kDa and 60 kDa in equal amounts. Like the DNA polymerase alpha isolated from eukaryotes, the alpha-like DNA polymerase possesses primase activity using UTP and polydeoxyadenylate as template. The primase activity was sensitive to aphidicolin and inhibited by an antiserum against the alpha-like DNA polymerase of H. halobium. The primase activity was dependent on the presence of high salt concentrations.

Antibodies↗