2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions.
All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes: E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1alpha, E1beta and E2 genes of the PDHC from gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed. (+info)
Haloterrigena thermotolerans sp. nov., a halophilic archaeon from Puerto Rico.
An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism, designated strain PR5T, is rod-shaped, non-motile and requires at least 12% (w/v) NaCl to grow. The strain is highly thermotolerant: its temperature optimum is 50 degrees C and growth is possible up to 60 degrees C. Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S2-DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. Both C20,C20 and C20,C25 core lipids are present. The G+C content of the DNA is 63.3 mol%. According to 16S rDNA sequence data, strain PR5T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5T cannot be assigned to any of the recognized species within these genera. On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena. The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate. The type strain is strain PR5T (= DSM 11552T = ATCC 700275T). (+info)
Extreme halophiles synthesize betaine from glycine by methylation.
Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles, Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochloris methyltransferase genes were successfully expressed in Escherichia coli, and betaine accumulation and improved salt tolerance were demonstrated. (+info)
Gene clusters encoding ATP synthase of Haloarcula japonica strain TR-1.
The genes encoding I, K, E, C, F, A, B and D subunits of A0A1 ATP synthase were cloned from Haloarcula japonica strain TR-1. The genes were split into two clusters. Northern analysis revealed that the larger gene cluster would be transcribed as a single mRNA. (+info)
The primary structures of helices A to G of three new bacteriorhodopsin-like retinal proteins.
The primary structures of helices A to G of all bacteriorhodopsin (BR)-like retinal proteins identified in newly isolated halobacteria have been determined from the nucleotide sequence of the BR-like protein genes. Using PCR methods, gene fragments encoding the A- to G-helix region of BR-like proteins were directly amplified from the total genomic DNA of the seven new halobacterial strains. Oligonucleotide primers corresponding to highly conserved regions in the helices A to G were designed from the nucleotide sequences of bacterioopsin (bop) and archaeopsin-I (aop-I), and some primers were effective for the amplification of the gene encoding C- to G-helix region of all new BR-like proteins. The primer corresponding to A-helix region was designed either from the nucleotide sequence of bop and aop-I or from the N-terminus amino acid sequence of a BR-like protein. Three new BR-like proteins were identified from the amino acid sequence, which was deduced from the nucleotide sequence of the genes encoding A- to G-helix region of the BR-like proteins. It was found that not only the amino acid sequence, but also the nucleotide sequence of the gene encoding the C- and G-helix region, in which a number of important residues for proton translocation are located, is highly conserved in three new BR-like proteins. Analysis of the primary structures of the A- to G-helix region of new BR-like proteins revealed that one has about 85% homology with aR-I and aR-II, and the rest have about 55% homology with halobium BR, aR-I and aR-II.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring.
The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems. (+info)
Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene.
Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations. (+info)
Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.
Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions. (+info)