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PubMed1996

Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles.

Gas vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the plasmid pHH1-encoded vac (p-vac) region consisting of 14 different gvp genes that are arranged in two clusters, p-gvpACNO and, oriented in the direction opposite to that of gvpA, p-gvpDEFGHIJKLM. The p-gvpACNO region was analyzed at the transcriptional and functional levels in H. salinarium and in Haloferax volcanii transformants containing subfragments of the p-vac region. The p-gvpACNO genes were transcribed as several mRNAs: the 270-nucleotide (nt) p-gvpA transcript, encoding the major structural protein, occurred in large amounts, and minor amounts of three different readthrough transcripts (p-gvpACN, and p-gvpACNO mRNA) were found. In addition, the p-gvpO gene gave rise to two separate mRNA species: a 550-nt mRNA starting at the ATG and spanning the entire reading frame and a 420-nt RNA encompassing the second half of the p-gvpO gene. The requirement of p-gvpC, p-gvpN, and p-gvpO gene expression for gas vesicle synthesis was assessed by transformation experiments using the VAC- species Haloferax volcanii as the recipient. A delta C transformant, harboring the p-vac region with a deletion of the p-gvpC gene, produced large amounts of irregularly shaped gas vesicles. A shape-forming function of p-GvpC was demonstrated by complementation of the delta C transformant with the p-gvpC gene, resulting in wild-type-shaped gas vesicles. In the delta N transformant, the level of gas vesicle synthesis was very low, indicating that the p-GvpN protein is not required for gas vesicle assembly but may enhance gas vesicle synthesis. The p-gvpN deletion did not affect accumulation of p-gvpACO mRNA but reduced the separate p-gvpO transcription. The delta O transformant was Vac- and had a strongly decreased level of p-gvpACN mRNAs, demonstrating that the p-GvpO protein is required for gas vesicle synthesis and may affect transcription of this DNA region.

Archaeal Proteins↗
PubMed1997

Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium.

Signal transduction in the archaeon Halobacterium salinarium is mediated by a family of 13 soluble and membrane-bound transducers. Here, we report the primary structure and functional analysis of one of the smallest halobacterial putative transducers, HtrXI. Hydropathy plot analysis of the primary structure predicts no membrane-spanning segments in HtrXI. The fractionation of the H. salinarium proteins confirmed that HtrXI is a soluble protein. Capillary assay with an HtrXI deletion mutant and a complemented strain revealed that this soluble transducer is involved in Asp and Glu taxis. In vivo analysis of the methylesterase activity of the htrXI-1 deletion mutant suggests that HtrXI plays an important role in the adaptation of the chemotactic responses to His, Asp, and Glu, which are attractants for halobacteria. Stimulation by Asp and Glu causes demethylation of HtrXI and of another putative transducer, HtrVII. But addition of His to halobacterial cells increases HtrXI methylation together with that of other putative transducers. In the absence of HtrXI, stimulation by either Glu or His does not decrease or increase the methylation of any putative transducers. Therefire, the HtrXI transducer appears to have a complex role in chemotaxis signal transduction.

Amino Acid Sequence↗
PubMed1999

Identification of methylation sites and effects of phototaxis stimuli on transducer methylation in Halobacterium salinarum.

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo L-[methyl-(3)H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained with Escherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited the E. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.

Amino Acid Sequence↗
PubMed2002

Identification of a lycopene beta-cyclase required for bacteriorhodopsin biogenesis in the archaeon Halobacterium salinarum.

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.

Amino Acid Sequence↗
PubMed2003

An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1.

We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.

Base Sequence↗
PubMed2005

Genomic analysis of anaerobic respiration in the archaeon Halobacterium sp. strain NRC-1: dimethyl sulfoxide and trimethylamine N-oxide as terminal electron acceptors.

We have investigated anaerobic respiration of the archaeal model organism Halobacterium sp. strain NRC-1 by using phenotypic and genetic analysis, bioinformatics, and transcriptome analysis. NRC-1 was found to grow on either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as the sole terminal electron acceptor, with a doubling time of 1 day. An operon, dmsREABCD, encoding a putative regulatory protein, DmsR, a molybdopterin oxidoreductase of the DMSO reductase family (DmsEABC), and a molecular chaperone (DmsD) was identified by bioinformatics and confirmed as a transcriptional unit by reverse transcriptase PCR analysis. dmsR, dmsA, and dmsD in-frame deletion mutants were individually constructed. Phenotypic analysis demonstrated that dmsR, dmsA, and dmsD are required for anaerobic respiration on DMSO and TMAO. The requirement for dmsR, whose predicted product contains a DNA-binding domain similar to that of the Bat family of activators (COG3413), indicated that it functions as an activator. A cysteine-rich domain was found in the dmsR gene, which may be involved in oxygen sensing. Microarray analysis using a whole-genome 60-mer oligonucleotide array showed that the dms operon is induced during anaerobic respiration. Comparison of dmsR+ and DeltadmsR strains by use of microarrays showed that the induction of the dmsEABCD operon is dependent on a functional dmsR gene, consistent with its action as a transcriptional activator. Our results clearly establish the genes required for anaerobic respiration using DMSO and TMAO in an archaeon for the first time.

Anaerobiosis↗
PubMed1967

Ultrastructure of the obligate halophilic bacterium Halobacterium halobium.

The fine structure of Halobacterium halobium was studied by means of a modified double-fixation technique. The cell envelope is shown to consist of both a "wall" and a plasma membrane. Some electron-dense strands were seen inside the cytoplasm running parallel to the cell envelope. An unusual organelle (or organelles) appeared inside the cytoplasm in the form of parallel striated strands.

Cell Wall↗
PubMed1969

Effect of salts and organic solvents on the activity of Halobacterium cutirubrum catalase.

Catalase in extracts of the extreme halophile Halobacterium cutirubrum exhibits up to threefold stimulation by 0.5 to 1.5 m monovalent salts and by 0.1 m divalent salts. Above these concentrations, inhibition of enzyme activity is observed. The inhibitory effect, and to some extent the stimulation, is salt-specific; the effectiveness of a salt in inhibiting enzyme activity depends on both cation and anion. Thus, the order of effectiveness is MgCl(2) > LiCl > NaCl > KCl > NH(4)Cl, and LiCl > LiNO(3) > Li(2)SO(4). The magnitude of enzyme inhibition for the salts tested is positively correlated with their molar vapor pressure depression in aqueous solution. Stimulation of enzyme activity was observed when one salt was added at its optimal concentration in the presence of inhibiting concentrations of another salt, indicating that the effect on the enzyme is not due to changing water activity but probably to enzyme-salt interaction. Aqueous solutions of ethylene glycol, glycerol, and dimethyl sulfoxide containing no ions influence enzyme activity in the same manner as do salts.

Ammonium Chloride↗
PubMed1976

Bacteriorhodopsin formation in Halobacterium halobium.

Systematic examinations were made of factors influencing bacteriorhodopsin formation during the growth of Halobacterium halobium. Light-induced adenosine triphosphate (ATP) production and [14C]proline uptake were used as measures of functional ability of the purple membrane. Maximum bacteriorhodopsin formation occurred under growth conditions of illumination and limited aeration. The purple membrane -ATP production system did not confer an appreciable growth advantage. Growth in the dark or with adequate aeration partially suppressed bacteriorhodopsin formation and the effects were additive. Nicotine effectively inhibited bacteriorhodopsin formation. A rapid synthesis of functional pigment occurred when washed suspensions of cells which had been grown under illumination with nicotine present were incubated under dark, aerobic conditions. The alleviation of this nicotine inhibition was not blocked by chloramphenicol or bacitracin. Bacteriorhodopsin formation was negligible when washed suspensions of cells from dark, limited aeration or light, adequate aeration cultures were incubated in the light with limited aeration. A nutritionally complex medium was needed to elicit appreciable bacteriorhodopsin formation by the cells from the dark or adequately aerated cultures. Bacitracin partially inhibited this bacteriorhodopsin formation by cells form the light, adequately aerated culture.

Adenosine Triphosphate↗
PubMed1978

The metabolism of carbohydrates by extremely halophilic bacteria: the identification of lactobionic acid as a product of lactose metabolism by Halobacterium saccharovorum.

Nongrowing cells of Halobacterium saccharovorum oxidized lactose to a product identified as lactobionic acid by thin-layer, paper, and column chromatography, and by identification of the galactose and gluconic acid produced from it after acid hydrolysis. Growing cells oxidized lactose to a product that was identical with lactobionate except that it did not serve as a substrate for galactose oxidase. While the identity of this compound has not been established, it is suggested that the product is lactobionic acid in which the galactose moeity is in the furanose form. Neither lactobionate nor the product produced by growing cells was further metabolized, suggesting that lactose oxidation is not coupled to growth.

Cells, Cultured↗
PubMed1979

Effect of glycerol on carotenogenesis in the extreme halophile, Halobacterium cutirubrum.

Growth of cells of Halobacterium cutirubrum in complex medium in the presence of 0.1 to 0.5% glycerol was slightly stimulated (about 10%), but the content of the major red pigment, bacterioruberin, was reduced fourfold and that of the minor red pigments, mono- and bisanhydrobacterioruberins, was also reduced but to a lesser extent. The content of the C40 carotenes, beta-carotene and lycopene, however, was greatly increased in 0.1 to 0.5% glycerol but was then reduced to negligible amounts at higher glycerol concentrations (1 to 5%). Similar effects of glycerol on carotenogenesis were observed when cells were grown in a chemically defined medium (BSMK), but growth of cells was considerably slower.

Carotenoids↗
PubMed1979

Studies of the biosynthesis of C50 carotenoids in Halobacterium cutirubrum.

Cells of Halobacterium cutirubrum were grown in the presence of [14C]mevalonate with and without 3 mM nicotine. Growth of cells was inhibited to a maximum extent of 25% but overall incorporation of 14C into total, neutral, or polar lipids was only inhibited about 16% during active growth and to a much lesser extent or not at all in stationary phase. Little effect of nicotine on labelling of squalenes, vitamin MK-8, geranylgeraniol, and phytoene was observed. However, labelling of bacterioruberin and monoanhydrobacterioruberin was extensively inhibited while that of lycopene and bisanhydrobacterioruberin was increased reciprocally by growth in the presence of 3 mM nicotine. Reincubation of the labelled nicotine-grown cells in fresh cold medium without nicotine restored the 14C-labelling of bacterioruberin and monoanhydrobacterioruberin at the expense of lycopene and bisanhydrobacterioruberin. These results confirm our previous findings suggesting that the C50 bacterioruberin is made by addition of a C5-isoprene unit to each end of the C40-lycopene chain, followed by introduction of four hydroxyl groups.

Carotenoids↗
PubMed1980

Exclusive formation of all-trans-phytoene by a colorless mutant of Halobacterium halobium.

A spontaneously occurring Halobacterium halobium mutant (strain W5002-1) was isolated which accumulated phytoene as the only C40 polyene. Its configuration was established as all-trans (7,8,11,12,7',8',11',12',-octahydro-psi,psi-carotene) by its chromatographic and spectral properties. Neither bacterioruberins (C50) nor retinal (C20) could be detected in this mutant strain. The biosynthetic pattern of isoprenoid compounds in this mutant strain confirms for the first time our conjecture that spontaneously arising H. halobium mutants can be isolated with defects in the carotenoid pathway and lends further support to our previous hypothesis suggesting all-trans-phytoene to be the precursor for more unsaturated carotenoids in halobacteria.

Carotenoids↗
PubMed1982

Bacteriophages of Halobacterium halobium: isolated from fermented fish sauce and primary characterization.

BActeriophages infecting extremely halophilic bacteria of the genus Halobacterium have been isolated from fermented anchovy sauce. Two distinct phages, designated Hh-1 and Hh-3, have been characterized. Both Hh-1 and Hh-3 are more tolerant of suspension in solutions of low ionic strength that their host bacteria. Both Hh-1 and Hh-3 have the ability to establish a carrier state upon infection of sensitive cells of H. halobium. Bacterial cells infected with phage in the carrier state are viable, produce phages, are immune to superinfection with homologous phages, yet remain fully capable of supporting heterologous phages. These properties suggest that the halophages are well adapted to survival in environments in which the salinity is subject to rapid changes of considerable magnitude.

Bacteriophages↗
PubMed1983

Bacteriophages of Halobacterium halobium: virion DNAs and proteins.

The DNAs from bacteriophages Hh-1 and Hh-3 that infect Halobacterium halobium were characterized. Both phages contain linear double-stranded DNA and show no relatedness based on restriction endonuclease fragment patterns. Hh-1 DNA has 67.05% guanine plus cytosine (G+C) and a molecular weight of 24.6 X 10(6), whereas Hh-3 DNA has 62.15% G+C and a molecular weight of 19.4 X 10(6). Polyacrylamide gel electrophoresis indicated that the major proteins of the two phages are of different molecular weights.

Bacteriophages↗
PubMed1989

Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations.

Several phenotypic variants of Halobacterium halobium arise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, the bop gene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes, gvpA and gvpC, are present on pNRC100, a multicopy plasmid of approximately 150 kilobase pairs. The gvpA and gvpC genes are on a single transcription unit, while the major bop mRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of the bop gene. Two elements, ISH 1 and ISH 2, account for most (80-90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number of gvpAC operon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗
PubMed1989

Isolation and partial characterization of plasmids found in three Halobacterium volcanii isolates.

Three new isolates of Halobacterium volcanii were screened for the presence of plasmids. Each of the different isolates was found to contain one plasmid. These plasmids do not show any homology to each other, nor to the previously isolated plasmid pHV2. Partial restriction maps of these plasmids were determined. One of the plasmids contains chromosomal repetitive sequences as judged by the existence of homologous sequences in the chromosomal DNA of the three isolates. Using the protoplast fusion technique, we showed that at least one of the newly isolated plasmids is compatible with pHV2.

Cloning, Molecular↗
PubMed1989

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants.

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.

Archaeal Proteins↗