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PubMed2000

The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.

Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.

Gene Expression Regulation, Archaeal↗
PubMed1992

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.

Anisomycin↗
PubMed1996

Isolation and chromosomal distribution of natural Z-DNA-forming sequences in Halobacterium halobium.

Conditions favoring left-handed Z-DNA such as high salinity (> 4 ), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG)n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5alpha with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.

Base Sequence↗
PubMed2000

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Bacteriorhodopsins↗
PubMed1986

ISH51: a large, degenerate family of insertion sequence-like elements in the genome of the archaebacterium, Halobacterium volcanii.

We describe a new family of repetitive elements in the genome of the archaebacterium Halobacterium volcanii. There are some 20-30 copies of this element, which we designate ISH51. Sequenced copies show typical insertion sequence characteristics (terminal inverted repeats, direct flanking repeats of "target site" DNA). However, members of the ISH51 family are highly heterogeneous, showing on average only 85% primary sequence homology; and some genomic copies appear to be severely truncated. Some ISH51 elements are clustered together in regions of relatively AT-rich DNA. There are at least five such AT-rich "islands" in the H. volcanii genome. Repetitive sequences homologous to ISH51 are found in the genomes of most Halobacterium and Halococcus species.

Base Sequence↗
PubMed1987

Effects of the antitumor drug VP16 (etoposide) on the archaebacterial Halobacterium GRB 1.7 kb plasmid in vivo.

The topoprofile of 1.7 kb plasmids from the archaebacterium Halobacterium GRB was analysed from cells growing with or without VP16 (etoposide). This drug interferes with the breakage-reunion reaction of eukaryotic DNA topoisomerase II by inhibiting the ligase activity of this enzyme. Addition of VP16 to the culture medium of Halobacterium GRB cells results in the introduction of single- and double-strand DNA breaks in part of the plasmid population, with proteins covalently associated at their 5' ends. While some of the remaining covalently closed circular DNA molecules are relaxed, VP16 treatment also gives rise to the production of positively supercoiled 1.7 kb plasmids. In contrast to adriamycin, VP16 does not intercalate into the 1.7 kb plasmid DNA in vivo. These results suggest that the VP16 target in halobacteria is a DNA topoisomerase II. Three major cleavage sites were detected on the 1.7 kb plasmid after VP16 treatment in vivo.

DNA, Bacterial↗
PubMed1989

Analysis of insertion mutants reveals two new genes in the pNRC100 gas vesicle gene cluster of Halobacterium halobium.

The archaebacterium, Halobacterium halobium, achieves buoyancy through synthesis of intracellular gas-filled vesicles. The plasmid-encoded gene (gvpA) specifying the major structural gas vesicle protein has previously been cloned and sequenced allowing the analysis of high-frequency mutations to the vesicle negative phenotype. Among eighteen gas vesicle mutants analyzed, four were observed to contain insertion elements 0.2 to 2 kb upstream of the structural gene. To explain the phenotype of these mutants, the upstream area was analyzed by DNA sequencing and transcriptional mapping. This analysis showed the presence of two open reading frames, gvpD and gvpE, which are of opposite transcriptional orientation to gvpA (gene order gvpA-D-E). gvpD begins 201 nucleotides from the gvpA structural gene and is 1608 nucleotides long while gvpE begins two nucleotides from the 3'-end of gvpD and is 573 nucleotides long. Primer extension analysis showed the occurrence of divergent promoters in the gvpA-gvpD intergenic region with the transcription start sites separated by 109 nucleotides. The sites of three insertion sequences in gas vesicle mutants mapped within gvpE while the fourth insertion site mapped near the N-terminal coding region of gvpD. Homology between the gvpDE gene region and a chromosomal site in a H. halobium NRC-1 derivative and in several other Halobacterium strains was identified by Southern hybridization.

Amino Acid Sequence↗
PubMed1994

Physical map and set of overlapping cosmid clones representing the genome of the archaeon Halobacterium sp. GRB.

We have constructed a complete, five-enzyme restriction map of the genome of the archaeon Halobacterium sp. GRB, based on a set of 84 overlapping cosmid clones. Fewer than 30 kbp, in three gaps, remain uncloned. The genome consists of five replicons: a chromosome (2038 kbp) and four plasmids (305, 90, 37, and 1.8 kbp). The genome of Halobacterium sp. GRB is similar in style to other halobacterial genomes by being partitioned among multiple replicons and by being mosaic in terms of nucleotide composition. It is unlike other halobacterial genomes, however, in lacking multicopy families of insertion sequences.

Chromosome Mapping↗
PubMed1989

Transfer of Halobacterium denitrificans (Tomlinson, Jahnke, and Hochstein) to the genus Haloferax as Haloferax denitrificans comb. nov.

Halobacterium denitrificans (Tomlinson, Jahnke, and Hochstein) was described at a time when the taxonomic subdivision of the family Halobacteriaceae was in a state of flux. On the basis of both biochemical and chemotaxonomic data, this organism exhibits features which indicate that it is more closely related to members of the genus Haloferax. On the basis of such criteria, we propose that Halobacterium denitrificans be reclassified as Haloferax denitrificans comb. nov. The type strain is strain ATCC 35960 (= DSM 4425).

Carbohydrate Metabolism↗
PubMed1999

Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov.

The 16S rRNA gene sequences of Halococcus saccharolyticus and Halococcus salifodinae were closely related (94.5-94.7% similarity) to that of Halococcus morrhuae, the type species of the genus Halococcus. However, Halococcus turkmenicus was distinct from the other members of this genus, with low 16S rRNA similarities when compared to Halococcus morrhuae (88.7%). On the basis of phylogenetic tree reconstruction, detection of signature bases and DNA-DNA hybridization data, it is proposed to transfer Halococcus turkmenicus to a novel genus, Haloterrigena, as Haloterrigena turkmenica gen. nov., comb. nov., and to accommodate Halobacterium trapanicum JCM 9743 and strain GSL-11 in the same species. On the basis of morphological, cultural and 16S rRNA sequence data, it is also proposed that the culture collection strains of Halobacterium trapanicum NCIMB 767, ATCC 43102 and JCM 8979 should be renamed as Halococcus sp.

Base Composition↗
PubMed2004

Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.

We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.

Animals↗
PubMed1988

Complete nucleotide sequence of the ribosomal 'A' protein operon from the archaebacterium, Halobacterium halobium.

DNA fragments cloned from the halophilic archaebacterium Halobacterium halobium including the gene for ribosomal 'A' protein (HhaL20) were sequenced. Sequence and transcript analyzes indicated that the 'A' protein message (ORFC) was co-transcribed with two neighbouring open reading frames (ORFA and B) as a single unit. Comparison of the amino acid sequences deduced from ORFA and B with those from other organisms demonstrated that ORFA encoded the protein homologous to the ribosomal protein L8 of Halobacterium cutirubrum (HcuL8) and L1 of Escherichia coli (EL1), while ORFB encoded the protein homologous with the human ribosomal protein P0. The sequences which were able to facilitate mRNA.16S-rRNA hybridization were found about 10-20 nucleotides upstream of the initiation codon ATG of each of the three ORFs. The 5'- and 3'-ends of the operon were mapped with nuclease S1. A putative promoter (A+T-rich) and termination signal (T-rich) were found to be present within the 5'- and 3'-flanking sequence.

Amino Acid Sequence↗
PubMed1979

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.

Adenosine Triphosphate↗
PubMed1993

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.

Amino Acid Sequence↗
PubMed1994

Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency.

Blotting, Southern↗
PubMed2004

Complexity of gas vesicle biogenesis in Halobacterium sp. strain NRC-1: identification of five new proteins.

The genome of Halobacterium sp. strain NRC-1 contains a large gene cluster, gvpMLKJIHGFEDACNO, that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additional gvp gene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-type Halobacterium sp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and beta-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.

Amino Acid Sequence↗
PubMed1966

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium.

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.

Adenine Nucleotides↗
PubMed1966

Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum.

Onishi, H. (National Research Council, Ottawa, Ontario, Canada), and D. J. Kushner. Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum. J. Bacteriol. 91:646-652. 1966.-Envelopes of Halobacterium cutirubrum dissolved rapidly in media of low ionic strength. Heating partially inhibited breakdown, probably because of nonspecific protein coagulation rather than inactivation of a lytic enzyme(s). Dissolution of envelopes in water did not involve splitting of peptide bonds or protein-lipid bonds, or any extensive breakdown of carbohydrate polymers. Dissolution was increased by alcohols and urea, even at high salt concentrations, but was not affected by metabolic inhibitors. Thus, no evidence was found for a dilution-activated lytic enzyme that contributes to envelope breakdown. Cells of H. cutirubrum were stable in 2 m NaCl, but lysis occurred in 2 m KCl or NH(4)Cl. This lysis did not involve an extensive breakdown of the envelope. No evidence for different sites of Na(+), K(+), and NH(4) (+) action was obtained from the pattern of release of envelope constituents in different concentrations of these salts. Ultracentrifugation studies showed that adding salts to envelopes that had been dissolved in water led to a nonspecific reaggregation of envelope material. No difference was seen between the effects of KCl and NaCl, except at 3 to 4 m concentrations where KCl caused more aggregation. The preferential effect of Na(+) on intact cells is probably due to its ability specifically to prevent leakage rather than to an overall effect on envelope integrity.

Alcohols↗