Halophilic Archaebacterium Halobacterium Research Articles

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum.

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH2-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS-PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS-PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.

Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin

We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.

Transcription of the halophage ΦH repressor gene is abolished by transcription from an inversely oriented lytic promoter

The temperate phage ΦH of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4. Rep acts at the transcriptional level by blocking the promoter for T4. The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage γ. Transcription of the rep gene does not occur when the phage is growing lytically. We show that this represson of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.

Antisense RNA mediates transcriptional processing in an archaebacterium, indicating a novel kind of RNase activity.

Strains of the extremely halophilic archaebacterium Halobacterium salinarium that are lysogenic for the phage phi H produce an antisense RNA transcript complementary to the first 151 nucleotides (nt) of the early lytic phage transcript T1. This is the first case of antisense control of gene expression in an archaebacterium. We show through transformation of H. salinarium that the antisense RNA functions in trans, rendering the early lytic phage transcript T1 susceptible to specific cleavage by an unidentified RNase of unique endonucleolytic activity. The single-stranded ends of RNA are cut off at the ends of the 151 nt RNA duplex, removing the ribosomal binding sites from the first open reading frame of transcript T1 but without concomitant digestion of the products.

Purification and properties of glutamine synthetase from the archaebacterium Halobacterium salinarium

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg2+ or Mn2+, the salt dependence was decreased and the thermostability increased. Mg2+ or Mn2+ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different Km-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn2+-and Mg2+-dependent activities at a different extent.

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.

Photoreceptors of halophilic archaebacterium phototaxis. Phoborhodopsin and sensory rhodopsin.

Extremely halophilic archaebacterium Halobacterium halobium cells show phototaxis to visible hight. The phototaxis requires vitamine A aldehyde, a retinal. Protein pigments containing retinal are the photoreceptors of the phototaxis. Phoborhodopsin (pR) is a photoreceptor of the negative phototaxis. Sensory rhodopsin (sR) is a photoreceptor of the positive phototaxis and S373, a transient photoproduct of sR, is another photoreceptor of the negative phototaxis. These photoreceptors are too unstable to isolate. Haloalkalophilic archaebacterium Natronobacterium pharaonis cells have pharaonis phoborhodopsin (ppR), which is also a photoreceptor of a negative phototaxis. PPR is solubilized and isolated in an active state, A methylation/demethylation system is involved in the signalling chain from photoreceptor to flagellar motor switch.

The gene for a halophilic glutamate dehydrogenase sequence, transcription analysis and phylogenetic implications

We have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5' end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.

Exonuclease activities associated with DNA polymerases α and β of the archaebacterium Halobacterium halobium

alpha-like and beta-like DNA polymerases have previously been isolated from a halophilic archaebacterium Halobacterium halobium. In this report, we show that the alpha-like DNA polymerase has an associated 3' to 5'-exonuclease activity which is specific for single-stranded DNA, sensitive to both aphidicolin and N-ethylmaleimide and dependent on high salt concentrations like the polymerase activity. As this DNA polymerase has been shown to contain a primase activity, it may be considered as the equivalent to both eukaryotic DNA polymerases alpha and delta. As shown by glycerol-gradient centrifugation and electrophoresis under denaturing conditions, the beta-like polymerase would appear to have a monomeric structure and comprise of a single 65-kDa polypeptide. This DNA polymerase has both 3' to 5'-exonuclease and 5' to 3'-exonuclease activities which, contrary to polymerase activity, are inhibited by high salt concentrations.

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Abstract— Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in vitro. Cells irradiated with ultraviolet (UV)‐fluences up to 350 J/m2 could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV‐irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non‐growth conditions did not influence the survival of UV‐irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV‐inactivation or photoreactivation. Small differences in the rates of UV‐inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400‐470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8‐hydroxy‐5‐deazaflavin type photoreactivating enzyme.

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.

Arginine deiminase from Halobacterium salinarium. Purification and properties

Arginine deiminase from the extreme halophilic archaebacterium Halobacterium salinarium was purified to homogeneity in a four-step procedure with a 310-fold enrichment. The enzyme consists of two identical subunits of 55 kDa; its native molecular mass is 105 kDa. The pI of 4.7 indicates that acidic nature of the protein, which is evidenced by its amino acid composition, which shows an excess of more than 15% of acidic amino acids. The N-terminal amino acid of the enzyme is lysine. Arginine deiminase from Halobacterium salinarium exhibits its highest catalytic activity in the presence of 3.5 M-NaCl, pH 7.6, and at 40 degrees C. The half-activity constant, Ks, for arginine is 3.1 mM. The enzyme is inhibited by ornithine.

Relationship of the membrane ATPase from Halobacterium saccharovorum to vacuolar ATPases

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan inhibited the halobacterial ATPase also in a nucleotide-protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuolar and the F-type ATPases.

Purification of ribosomal proteins from the extreme halophilic archaebacteriumHalobacterium marismortui by high-performance liquid chromatography

High performance liquid chromatography was used for the purification of ribosomal proteins derived from the halophilic archaebacteriumHalobacterium marismortui. Separation was performed using size exclusion chromatography, reversed phase and ion-exchange chromatography. For reversed phase separation several solvent systems were te sted including isopropanol and acetonitrile. Tris/citrate buffer containing 30% N,N-dimethylformanide was employed in combination with a DEAE-anoin-exchanger.

Unusual evolution of a superoxide dismutase-like gene from the extremely halophilic archaebacterium Halobacterium cutirubrum

The archaebacterium Halobacterium cutirubrum contains a single detectable, Mn-containing superoxide dismutase, which is encoded by the sod gene (B. P. May and P. P. Dennis, J. Biol. Chem. 264:12253-12258, 1989). The genome of H. cutirubrum also contains a closely related sod-like gene (slg) of unknown function that has a pattern of expression different from that of sod. The four amino acid residues that bind the Mn atom are conserved, but the flanking regions of the two genes are unrelated. Although the genes have 87% nucleotide sequence identity, the proteins they encode have only 83% amino acid sequence identity. Mutations occur randomly at the first, second, and third codon positions, and transversions outnumber transitions. Most of the mutational differences between the two genes are confined to two limited regions; other regions totally lack differences. These two gene sequences are apparently in the initial stage of divergent evolution. Presumably, this divergence is being driven by strong selection at the molecular level for either acquisition of new functions or partition and refinement of ancestral functions in one or both of the respective gene products.

Transcriptional induction of purple membrane and gas vesicle synthesis in the archaebacterium Halobacterium halobium is blocked by a DNA gyrase inhibitor

We have investigated the expression of the bacteriorhodopsin gene (bop) and the gas vesicle protein gene (gvpA) in the extremely halophilic archaebacterium Halobacterium halobium, using primer-directed reverse transcription of RNA to quantify message levels. The level of gvpA gene transcript was found to increase about 5-fold from early to mid-logarithmic growth phase, while the level of bop gene transcript increased about 20-fold from mid-logarithmic to stationary phase. Transcriptional induction of both the gvpA and bop genes was significantly reduced by aeration and almost completely blocked by the DNA gyrase inhibitor novobiocin.

A Comparative Study of the Lipid Composition of Halobacterium saccharovorum from Various Sources

Summary Investigations on the taxonomic status of the extremely halophilic archaebacterium Halobacterium saccharovorum have shown that it is deserving of separate species status, but that there is some dispute as to its exact position within the taxonomy of the family Halobacteriaceae . In order to clarify the position of H. saccharovorum within the family Halobacteriaceae a chemotaxonomic study of this species was undertaken using strains obtained from five different sources. This work shows that one of the strains is anomalous and should not be included in the species H. saccharovorum .

Functional implications related to the gene structure of the elongation factor EF-Tu fromHalobacterium 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.

Lactate Dehydrogenase from the Extreme Halophilic ArchaebacteriumHalobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii: The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.