Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.
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Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya. (+info)
Bioenergetic aspects of halophilism.
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Examination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H2 + CO2 or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic Archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA+ and K+ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H2 + CO2 exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not. (+info)
Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt).
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The motile, predominantly square-shaped, red archaeon strain 801030/1T, isolated from a brine pool in the Sinai peninsula (Egypt), was characterized taxonomically. On the basis of its polar lipid composition, the nucleotide sequences of its two 16S rRNA genes, the DNA G+C content (60.1 mol%) and its growth characteristics, the isolate could be assigned to the genus Haloarcula. However, phylogenetic analysis of the two 16S rRNA genes detected in this organism and low DNA-DNA hybridization values with related Haloarcula species showed that strain 801030/1T is sufficiently different from the recognized Haloarcula species to warrant its designation as a new species. A new species, Haloarcula quadrata, is therefore proposed, with strain 801030/1T (= DSM 11927T) as the type strain. (+info)
An improved transposon for the halophilic archaeon Haloarcula hispanica.
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An improved transposon (ThD73) for Haloarcula hispanica is described. Based on the halobacterial insertion sequence ISH28, it showed little target sequence specificity but was biased toward a lower G+C content. Twenty randomly selected ThD73 mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements. (+info)
Transcriptional regulation of cruxrhodopsin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.
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Transcription of the cruxrhodopsin (cR) gene in extremely halophilic archaeon Haloarcula japonica strain TR-1 was investigated using Northern analysis to quantify message level. In the cell cultures growing in the dark, cR transcript level remained very low. In contrast, exposure of the cell cultures to light stimulated transcription of the cR gene. In addition, cR gene transcription was also induced when Ha. japonica was grown under high oxygen tension and then shifted to low oxygen tension in the dark. These results suggested that transcription of the cR gene is regulated by high light intensity and low oxygen tension. (+info)
Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.
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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. (+info)
A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2.
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The mevinolin-resistance determinant, hmg, encodes the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and is a commonly used selectable marker in halobacterial genetics. Plasmids bearing this marker suffer from instability in Haloferax volcanii because the resistance gene was derived from the genome of this species and is almost identical in sequence to the chromosomal copy. In order to reduce the level of homologous recombination between introduced plasmid vectors and the chromosome of Haloferax, a homologue of the hmg determinant was obtained from the distantly related organism, Haloarcula hispanica. The nucleotide sequences of the wild-type genes (hmgA) of these two species are only 78% identical, and the predicted protein sequences show 71% identity. In comparison to the wild-type hmgA gene, the resistance gene from a mutant resistant to simvastatin (an analogue of mevinolin) showed a single base substitution in the putative promoter. Plasmids constructed using the new resistance determinant were stably maintained under selection in Hfx. volcanii and possessed very low recombination rates with the chromosome of this species. In addition, an improved strain of Hfx. volcanii was developed to overcome the plasmid instability and growth reduction observed in the commonly used WFD11 strain. (+info)
Structural insights into peptide bond formation.
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The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-A resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid-biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The alpha-NH(2) group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2' OH of the A-76 ribose in the P site or with the 2' OH of A2486 (2451). These interactions position the alpha amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack. (+info)