Folic acid and pteroylpolyglutamate contents of archaebacteria.

Cell extracts of methanogens and the thermoacidophile Sulfolobus solfataricus contained little or no folic acid (pteroylglutamate) or pteroylpolyglutamate activity (less than 0.1 nmol/g [dry weight]). However, the halophile Halobacterium salinarum contained pteroylmono- or pteroyldiglutamates, and Halobacterium volcanii and Halobacterium halobium contained pteroyltriglutamates at levels equivalent to those in eubacteria (greater than 1 nmol/g [dry weight]).     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.     

Three-dimensional structure of the regular surface glycoprotein layer of Halobacterium volcanii from the Dead Sea

A three-dimensional reconstruction from electron micrographs of negatively stained cell envelopes of Halobacterium volcanii has revealed the structure of the surface glycoprotein to a resolution of 2 nm. The glycoprotein is arranged on a p6 lattice with a lattice constant of 16.8 nm. It forms 4.5 nm high, dome-shaped, morphological complexes with a narrow pore at the apex opening into a `funnel' towards the cell membrane. The polarity of the structure was derived from freeze-etching experiments and `edge' views. Six radial protrusions emanate from each morphological complex and join around the 3-fold axis to provide lateral connectivity. Using the primary structure of the surface glycoprotein of the closely related species Halobacterium halobium (Lechner and Sumper, 1987) and the cell envelope profile from a previous X-ray analysis of the same species (Blaurock et al., 1976) we have integrated our reconstruction into a model of halobacterial cell envelope.     

Isolation and characterization of dolichol-linked oligosaccharides from Haloferax volcanii

Biosynthesis of procaryotic glycoproteins has been studied insome detail only in the extreme halophile Halobacterium halobium.To extend these studies for a moderate halophile, dolichol phosphate-linkedoligosaccharides were isolated and characterized from Haloferaxvolcanii. Mannosyl (ß1     

Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively. The deduced amino acid sequences of the A- and B-subunits of Ha. japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii. The consensus ATP-binding motif was found in the A-subunit.     

Orientation of porin channels in the outer membrane of Bordetella pertussis

We have examined the surface topography and channel connectivity of a naturally crystalline porin that is known to be functional, and whose structure has not been perturbed by detergent extraction, A three-dimensional density map, calculated from two independent tilt series of negatively stained cell envelopes, reveals three separate channels per trimer on one side (the ‘smooth’ side), and a single common opening at the other (‘rough’) side. This arrangement is consistent with the molecular structures recently determined at high resolution by X-ray crystallography for three other porins after detergent solubilization, and implies that the Bordetella pertussis porin may have the same kind of folding. Surface relief maps calculated from electron micrographs of cell envelopes contrasted by unidirectional shadowing clearly show that the side with single opening (i.e. the rough side) represents the external surface.     

From replication to cultivation: hot news from Haloarchaea

Haloarchaea have developed into model organisms that are utilized to study many biological processes. Examples are the mechanisms of chromosome maintenance, gene expression and its regulation, protein export and degradation, and motility and sensing. In addition to the analysis of model species like Halobacterium salinarum and Haloferax volcanii, natural communities have been characterized. Halophilic Archaea were found in low-salt environments and are thus more widespread than previously thought.     

A comparison of liquid-holding recovery and photoreactivation in halophilic and non-halophilic bacteria

The ability of the extreme halophile Halobacterium cutirubrum to recover from the effects of ultraviolet radiation during liquid holding in the dark in non-nutrient medium has been compared with that of (i) a moderately halophilic bacterium (NRC 41227) and (ii) Escherichia coli B. The photoreactivabilities of all three bacteria have also been studied. The extreme halophile was incapable of liquid-holding recovery in these conditions, in marked contrast to both E. coli B and the moderate halophile, and also failed to recover when held in nutrient medium in the dark. These results strongly support the hypothesis that H. cutirubrum lacks DNA excision repair. It was also found that ultraviolet-irradiated H. cutirubrum could be almost completely photoreactivated from any level of survival in the range 10(-4)-80%, provided exposure to visible light was not delayed, whereas the moderate halophile resembled E. coli B and had a comparatively limited capacity for photoreactivation.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

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.     

Aminoacylation of an unusual tRNA(Cys) from an extreme halophile

The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein-nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase. Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts. This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution. Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core. This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core.     

8-Hydroxy-5-deazaflavin-dependent electron transfer in the extreme halophile Halobacterium cutirubrum

Abstract Cell extracts of the extreme halophile Halobacterium cutirubrum were found to contain 8-hydroxy-5-deazaflavin as well as 8-OH-5-deazaflavin: NADPH oxidoreductase activity. The oxidoreductase was partially purified and showed maximum activity at pH 5.4, which is unusually low for halobacteria, and 5.3 M NaCl, close to the intracellular salt concentration. The results indicate the presence of an 8-OH-5-deazaflavin-dependent electron transfer system in a nonmethanogenic organism.     

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).     

An 8-A projected structure of FhuA, A "ligand-gated" channel of the Escherichia coli outer membrane.

The structure of FhuA, a siderophore and phage receptor in the outer membrane of Escherichia coli, has been investigated by electron crystallography. Bidimensional crystals of hexahistidine-tagged FhuA protein solubilized in N,N-dimethyldodecylamine-N-oxide were produced after detergent removal with polystyrene beads. Frozen-hydrated crystals (unit cell dimensions of a = 124 A, b = 98 A, gamma = 90 degrees ) exhibited a p22121 plane group symmetry. A projection map at 8 A resolution showed the presence of dimeric ring-like structures with an elliptical shape (48 x 40 A). Each monomer was composed of a ring of densities with a radial width of 8-10 A corresponding to a cylinder of beta sheets. Few densities are present inside the barrel, leaving a central channel approximately 25 A in diameter. A projection map of FhuA at 15 A resolution, which was calculated from negatively stained preparations, demonstrated that most of the central channel was masked by extramembrane domains. This map also revealed an asymmetric distribution of extramembrane domains in FhuA, with large domains located mainly on one side of the molecule. Comparison with density maps derived from recent atomic structure allowed further interpretation of the electron microscopy projection structures with regard to long hydrophilic loops governing the selectivity and opening of the channel.