Conserved sequence elements involved in regulation of ribosomal protein gene expression in halophilic archaea.
ABSTRACT: A region of the Haloferax volcanii genome encoding ribosomal proteins L11e, L1e, L10e, and L12e was cloned and sequenced, and the transcripts derived from the cluster were characterized. Flanking and noncoding regions of the sequence were analyzed phylogenetically by comparison with the homologous sequences from two other halophilic archaea, i.e., Halobacterium cutirubrum and Haloarcula marismortui. Motifs, identified by high-level sequence conservation, include both transcriptional and translational regulatory elements and other elements of unknown function.
Project description:Four species representing three genera of halophilic archaebacteria were examined for the presence of genomic sequences that encode proteins of the superoxide dismutase family. Three species, Halobacterium cutirubrum, Halobacterium sp. strain GRB, and Haloferax volcanii, contain duplicated (paralogous) genes of the sod family; a fourth species, Haloarcula marismortui, contains only a single gene. These seven genes were cloned and sequenced, and their transcripts were characterized by Northern (RNA) hybridization, S1 nuclease protection, and primer extension. The expression of one of the two genes in H. cutirubrum, Halobacterium sp. strain GRB, and Haloferax volcanii was shown to be elevated in the presence of paraquat, a generator of superoxide radicals. The other genes, including the single gene from Haloarcula marismortui, exhibited no elevated expression in the presence of paraquat. The 5' and 3' flanking regions of all the genes contain recognizable promoter and terminator elements that are appropriately positioned relative to the 5' and 3' transcript end sites. Between genera, the orthologous paraquat-responsive genes exhibit no sequence similarity in either their 5' or 3' flanking regions, whereas the orthologous nonresponsive genes exhibit limited sequence similarity but only in the 5' flanking region. Within the coding region, the two paralogous genes of Haloferax volcanii are virtually identical (99.5%) despite the absence of similarity in the flanking regions. In contrast, the paralogous genes of H. cutirubrum and Halobacterium sp. strain GRB are only about 87% identical. In the alignment of all seven sequences, there are nine codon positions where both the TCN and AGY serine codons are utilized; some or all of these may well be examples of convergent evolution.
Project description:In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature.
Project description:Halophilic archaea often inhabit environments with limited oxygen, and many produce ion-pumping rhodopsin complexes that allow them to maintain electrochemical gradients when aerobic respiration is inhibited. Rhodopsins require a protein, an opsin, and an organic cofactor, retinal. We previously demonstrated that in Halobacterium salinarum, bacterioopsin (BO), when not bound by retinal, inhibits the production of bacterioruberin, a biochemical pathway that shares intermediates with retinal biosynthesis. In this work, we used heterologous expression in a related halophilic archaeon, Haloferax volcanii, to demonstrate that BO is sufficient to inhibit bacterioruberin synthesis catalyzed by the H. salinarum lycopene elongase (Lye) enzyme. This inhibition was observed both in liquid culture and in a novel colorimetric assay to quantify bacterioruberin abundance based on the colony color. Addition of retinal to convert BO to the bacteriorhodopsin complex resulted in a partial rescue of bacterioruberin production. To explore if this regulatory mechanism occurs in other organisms, we expressed a Lye homolog and an opsin from Haloarcula vallismortis in H. volcaniiH. vallismortis cruxopsin-3 expression inhibited bacterioruberin synthesis catalyzed by H. vallismortis Lye but had no effect when bacterioruberin synthesis was catalyzed by H. salinarum or H. volcanii Lye. Conversely, H. salinarum BO did not inhibit H. vallismortis Lye activity. Together, our data suggest that opsin-mediated inhibition of Lye is potentially widespread and represents an elegant regulatory mechanism that allows organisms to efficiently utilize ion-pumping rhodopsins obtained through lateral gene transfer.IMPORTANCE Many enzymes are complexes of proteins and nonprotein organic molecules called cofactors. To ensure efficient formation of functional complexes, organisms must regulate the production of proteins and cofactors. To study this regulation, we used bacteriorhodopsin from the archaeon Halobacterium salinarum Bacteriorhodopsin consists of the bacterioopsin protein and a retinal cofactor. In this article, we further characterize a novel regulatory mechanism in which bacterioopsin promotes retinal production by inhibiting a reaction that consumes lycopene, a retinal precursor. By expressing H. salinarum genes in a different organism, Haloferax volcanii, we demonstrated that bacterioopsin alone is sufficient for this inhibition. We also found that an opsin from Haloarcula vallismortis has inhibitory activity, suggesting that this regulatory mechanism might be found in other organisms.
Project description:The genomes of two extremely halophilic Archaea species, Haloarcula marismortui and Haloferax mediterranei, were sequenced using single-molecule real-time sequencing. The ?4-Mbp genomes are GC rich with multiple large plasmids and two 4-methyl-cytosine patterns. Methyl transferases were incorporated into the Restriction Enzymes Database (REBASE), and gene annotation was incorporated into the Haloarchaeal Genomes Database (HaloWeb).
Project description:Recent insight into the N-glycosylation pathway of the haloarchaeon, Haloferax volcanii, is helping to bridge the gap between our limited understanding of the archaeal version of this universal post-translational modification and the better-described eukaryal and bacterial processes. To delineate as yet undefined steps of the Hfx. volcanii N-glycosylation pathway, a comparative approach was taken with the initial characterization of N-glycosylation in Haloarcula marismortui, a second haloarchaeon also originating from the Dead Sea. While both species decorate the reporter glycoprotein, the S-layer glycoprotein, with the same N-linked pentasaccharide and employ dolichol phosphate as lipid glycan carrier, species-specific differences in the two N-glycosylation pathways exist. Specifically, Har. marismortui first assembles the complete pentasaccharide on dolichol phosphate and only then transfers the glycan to the target protein, as in the bacterial N-glycosylation pathway. In contrast, Hfx. volcanii initially transfers the first four pentasaccharide subunits from a common dolichol phosphate carrier to the target protein and only then delivers the final pentasaccharide subunit from a distinct dolichol phosphate to the N-linked tetrasaccharide, reminiscent of what occurs in eukaryal N-glycosylation. This study further indicates the extraordinary diversity of N-glycosylation pathways in Archaea, as compared with the relatively conserved parallel processes in Eukarya and Bacteria.
Project description:We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea. The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62%. Comparison of the genome architectures of Halobacterium sp. NRC-1 and H. marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former. Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm. A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms. Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp. NRC-1--suggesting H. marismortui is significantly more physiologically capable of exploiting diverse environments. In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile. Finally, as expected from the larger genome, H. marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp. NRC-1.
Project description:Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of gamma-glutamylcysteine (gamma GC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of gamma GC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.
Project description:Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform.
Project description:The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 of Halobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants. The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H. volcanii. The gas vesicles formed in some of these gvp gene deletion transformants were altered in shape (Delta I, Delta C) or strength (Delta H) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H. volcanii. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed only gvpFG(J), suggesting that the gvpJKLM genes were not sufficiently expressed. Since gvpAO and gvpFGJKLM are the only gvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterial gvp genes are present in Bacillus megaterium and in the genome of Streptomyces coelicolor.
Project description:Genome-wide analysis has revealed abundant FabG (beta-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the DeltafabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.