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PubMed2006

Proteome analysis of Halobacterium sp. NRC-1 facilitated by the biomodule analysis tool BMSorter.

To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms.

Amino Acids↗
PubMed1975

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.

Cell Fractionation↗
PubMed1996

Organization and nucleotide sequences of ten ribosomal protein genes from the region equivalent to the S10 operon in the archaebacterium, Halobacterium halobium.

A determination was made of the nucleotide sequence of the 7340-bp region of a ribosomal protein gene cluster of Halobacterium halobium, which is equivalent to the S10 operon of Escherichia coli. The sequence was analyzed with the codonpreference program deduced from the halobacterial codon usage table that showed a very high GC content of the third codon position. The sequence was comprised of a string of 13 tightly linked ORFs. Most of the ORFs were homologous with ribosomal protein genes (ORF1-ORF2-rpl3-rpl4-rpl23--rpl2- rps19-rpl22-rps3-rpl29-ORF11-rps17-r pl14). The 13-gene string was preceded by three putative AT-rich promoter sequences. The order of the genes in H. halobium essentially agreed with that of the corresponding genes of E. coli (S10-operon), except for certain deletions or insertions of additional protein genes.

Amino Acid Sequence↗
PubMed1998

Cloning and sequencing of the homologues of both the bacterial and eukaryotic initiation factor genes (hIF-2 and heIF-2 gamma) from archaeal Halobacterium halobium.

The cloning and sequencing of the genes encoding the translational initiation factors (hIF-2 and heIF-2 gamma) was performed by screening the halophilic archaeon Halobacterium halobium genomic library with a probe constructed from the peptide IGHVDHGK that is conserved in archaeal GTP-binding elongation factors. The codon usage by the hIF-2 and heIF-2 gamma genes showed a preference for triplets ending in G or C. This characteristic is almost identical to that of other H. halobium genes. The translated protein of hIF-2 and heIF-2 gamma genes is made of 414 and 583 amino acid residues, respectively, and contains the sequence motif for the binding of GTP. The sequence of hIF-2 shows a strong similarity to the initiation factor IF-2 from Bacteria whereas heIF-2 gamma shows a strong similarity to the initiation factor eIF-2 gamma from Eucarya.

Amino Acid Sequence↗
PubMed1968

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes.

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.

Amino Acids↗
PubMed1976

Structure of the cell envelope of Halobacterium halobium.

The structure of the isolated cell envelope of Halobacterium halobium is studied by X-ray diffraction, electron microscopy, and biochemical analysis. The envelope consists of the cell membrane and two layers of protein outside. The outer layer of protein shows a regular arrangement of the protein or glycoprotein particles and is therefore identified as the cell wall. Just outside the cell membrane is a 20 A-thick layer of protein. It is a third structure in the envelope, the function of which may be distinct from that of the cell membrane and the cell wall. This inner layer of protein is separated from the outer protein layer by a 65 A-wide space which has an electron density very close to that of the suspending medium, and which can be etched after freeze-fracture. The space is tentatively identified as the periplasmic space. At NaCl concentrations below 2.0 M, both protein layers of the envelope disintegrate. Gel filtration and analytical ultracentrifugation of the soluble components from the two protein layers reveal two major bands of protein with apparent mol wt of approximately 16,000 and 21,000. At the same time, the cell membrane stays essentially intact as long as the Mg++ concentration is kept at treater than or equal to 20 mM. The cell membrane breaks into small fragments when treated with 0.1 M NaCl and EDTA, or with distilled water, and some soluble proteins, including flavins and cytochromes, are released. The cell membrane apparently has an asymmetric core of the lipid bilayer.

Bacterial Proteins↗
PubMed1978

Bacteriorhodopsin induces a light-scattering change in Halobacterium halobium.

When suspensions of Halobacterium halobium are exposed to bright light, the light-scattering properties of the bacteria change. This light-scattering response can produce a transmission decrease of about 1% throughout the red and near-infrared region. The action spectrum for the light-scattering response appropriately matches the absorption spectrum of bacteriorhodopsin. The response is eliminated by cyanide p-trifluoro-methoxyphenylhydrazone, a proton ionophore, and by triphenylmethylphosphonium, a membrane permanent cation. A mild hypertonic shock induces a similar light-scattering change, suggesting that bright light causes the bacteria to shrink about 1% in volume, thereby producing the light-scattering response.

Adaptation, Physiological↗
PubMed1970

Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations.

Work is described on the changes in cell ions during growth of cultures of a species of Halobacterium isolated from the Dead Sea. Cell K concentration fell from 5.5 to 3.8 moles per kg cell water during the logarithmic phase of growth and maintained the latter value during the stationary phase (initial medium concentration, 7 mM). Cell Na and Cl followed a complex series of roughly parallel changes. The logarithmic phase ion concentrations were: Na, 1.0-2.3 moles/kg cell water; Cl, 2.3-3.7 moles/kg cell water. The final stationary phase values were: Na, 0.5 moles/kg cell water; Cl, 2.3-2.9 moles/kg cell water (medium NaCl concentration, 3.9 Molal). It is suggested that most of the K(+) is bound within the cytoplasm.

Centrifugation↗
PubMed1999

Car: a cytoplasmic sensor responsible for arginine chemotaxis in the archaeon Halobacterium salinarum.

A new metabolic signaling pathway for arginine, both a chemoeffector and a fermentative energy source, is described for Halobacterium salinarum. Systematic screening of 80+ potentially chemotactic compounds with two behavioral assays identified leucine, isoleucine, valine, methionine, cysteine, arginine and several peptides as strong chemoattractants. Deletion analysis of a number of potential halobacterial transducer genes led to the identification of Car, a specific cytoplasmic arginine transducer which lacks transmembrane helices and was biochemically shown to be localized in the cytoplasm. Flow assays were used to show specific adaptive responses to arginine and ornithine in wild-type but not Deltacar cells, demonstrating the role of Car in sensing arginine. The signaling pathway from external arginine to the flagellar motor of the cell involves an arginine:ornithine antiporter which was quantitatively characterized for its transport kinetics and inhibitors. By compiling the chemotactic behavior, the adaptive responses and the characteristics of the arginine:ornithine antiporter to arginine and its analogs, we now understand how the combination of arginine uptake and its metabolic conversion is required to build an effective sensing system. In both bacteria and the archaea this is the first chemoeffector molecule of a soluble methylatable transducer to be identified.

Amino Acids↗
PubMed1998

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium.

The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N-glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser-, in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N-glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.

Archaeal Proteins↗
PubMed1984

Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium.

Base Composition↗
PubMed1985

Common structural features of the genes for two stable RNAs from Halobacterium halobium.

The genes coding for the 5S rRNA and another stable RNA, termed 7S RNA, in Halobacterium halobium were isolated from a genomic library of this archaebacterium and their nucleotide sequences determined. Both genes are colinear with their transcripts (5S rRNA and 7S RNA), but 5S rRNA and possibly also 7S RNA isolated from other halobacteria carry additional nucleotides within the RNA transcript. Both genes are located in the G + C rich chromosomal fraction I of H. halobium. Comparison of the 3' non-coding regions of both genes shows a 20 bp sequence of high homology immediately at the 3' ends which is almost symmetrically flanked by two stem-loop structures, one being situated close to the 3' end but within the coding region and the other downstream of the common 20 bp sequence.

Base Sequence↗
PubMed1988

Novobiocin induces accumulation of a single strand of plasmid pGRB-1 in the archaebacterium Halobacterium GRB.

Treatment of Halobacterium GRB cells with the DNA topoisomerase II inhibitor novobiocin induces the accumulation of a circular single-stranded DNA form of the plasmid pGRB-1. This form corresponds to the transcribed strand of pGRB-1. A tiny amount of this form is detectable in untreated cells. The induction of single-stranded pGRB-1 molecules by novobiocin is abolished when cells are pretreated with aphidicolin or anisomycin, which inhibit halobacterial DNA replication and protein synthesis, respectively. These results suggest that the single-stranded form of pGRB-1 is generated in the course of plasmid replication.

Bacterial Proteins↗
PubMed1990

Transposition burst of the ISH27 insertion element family in Halobacterium halobium.

Investigation of the plasmid pHH4 in single colonies of Halobacterium halobium PHH4 indicated transposition of insertion elements in 20% of the colonies. Seven ISH27 insertions were observed as well as one ISH23 insertion. The various copies of ISH27 were compared to the two ISH27 elements already present in pHH4, and to the ISH27 element that was identified in the bacteriopsin (bop) gene of a Bop mutant. These ten copies of ISH27 constitute three types on the basis of DNA sequence identity: ISH27-1 (1398 bp), ISH27-2, and ISH27-3 (1389 bp each). The DNA sequence comparison between the three types indicates a region of 1200 bp where the identity between ISH27-1 and ISH27-2 or ISH27-3 is 82-83%. ISH27-2 and ISH27-3 are 95% identical in this region. The remaining region exhibits a lower DNA similarity (64-74% identity) between the different copies. An open reading frame of 1167 nucleotides spans the more conserved region, and a corresponding transcript could be detected in H. halobium PHH4, but not in H. halobium wild-type. ISH27-1 is 91% identical to members of the insertion sequence-like elements ISH51 of Haloferax volcanii, whereas the other two ISH27 element types are 82-83% identical to ISH51. The transposition 'burst' of ISH27 was only seen after storage of the cells for more than two years at 4 degrees C. Upon continuous cultivation at 37 degrees C no transposition event could be observed, suggesting that stress factor(s) might have caused the high transposition rate.

Amino Acid Sequence↗
PubMed1990

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.

Amino Acid Sequence↗
PubMed1993

Structure specific ds/ss-RNase activity in the extreme halophile Halobacterium salinarium.

A ds/ss-RNA processing activity involved in antisense-RNA mediated gene regulation in the extremely halophilic archaebacterium Halobacterium salinarium was investigated in vivo. H.salinarium cells were transformed with DNA encoding an RNA species complementary to a part of the major lytic transcript, termed T4, of the H.salinarium phage phi H. The transformants transcribing this construct, when infected by phage were able to process T4 in a similar way to the processing of the lytic transcript denoted T1, in the natural sense-antisense system. Processing of T4 was not observed under normal phage growth on wild-type cells. Thus the antisense-RNA mediated processing activity earlier reported is dependent on the presence of an RNA duplex and is not sequence specific.

Bacteriophages↗
PubMed1979

The number, physical organization and transcription of ribosomal RNA cistrons in an archaebacterium: Halobacterium halobium.

Because it is now clear that archaebacteria may be as distinct from eubacteria as either group is from eukaryotic cells, and because a specifically archaebacterial ancestry has been proposed for the nuclear-cytoplasmic component of eukaryotic cells, we undertook to characterize, for the first time, the ribosomal RNA cistrons of an archaebacterium (Halobacterium halobium). We found these cistrons to be physically linked in the order 16S-23S-5S, and obtained evidence that they are also transcribed from a common promoter(s) in the order 5'-16S-23S-5S-3'. We showed that, although slightly larger immediate precursors of 16S and 23S are readily seen, no common precursor of both 16S and 23S can be easily detected in vivo. In all these respects the archaebacterium H. halobium is like a eubacterium and unlike the nuclear-cytoplasmic component of eukaryotic cells. We found, however, that it differs from eubacteria of comparable (large) genome size in having only one copy of the rRNA gene cluster per genome.

DNA, Bacterial↗
PubMed1998

A two-dimensional 1H detected 13C NMR investigation of pyruvate metabolism in Halobacterium salinarium.

Two-dimensional 1H detected 13C NMR spectroscopy has been used to study the intracellular metabolism of [3-(13)C]pyruvate in Halobacterium salinarium. The method, resulting in considerable improvement in spectral resolution and signal-to-noise ratio, is well suited for studying transient metabolic intermediates. Pyruvate utilization by the bacterium is a double exponential function with rate constants of 49.13 and 4.67x10(-3) per min. The relative 13C enrichment is the fastest for C-3 glutamate. Glutamate C-4 labeling decreases initially and increases later on during incubation, while glutamine C-3 is high to begin with and exhibits a declining trend. The glutamate labeling indicates a high initial flux through pyruvate carboxylase and extensive randomizing of the label in the tricarboxylic acid cycle.

Citric Acid Cycle↗