GlpR functions as a global DeoR-type regulator of metabolic gene transcription in the haloarchaeon Haloferax volcanii
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ABSTRACT:
ORGANISM(S): Haloferax volcanii
PROVIDER: GSE77589 | GEO | 2017/02/01
SECONDARY ACCESSION(S): PRJNA310954
REPOSITORIES: GEO
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Project description:Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.
| S-EPMC2676566 | biostudies-literature
Project description:The extremely halophilic archaeon Haloferax volcanii grows anaerobically by denitrification. A putative DNA-binding protein, NarO, is encoded upstream of the respiratory nitrate reductase gene of H. volcanii. Disruption of the narO gene resulted in a loss of denitrifying growth of H. volcanii, and the expression of the recombinant NarO recovered the denitrification capacity. A novel CXnCXCX7C motif showing no remarkable similarities with known sequences was conserved in the N terminus of the NarO homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved by expression of any mutant NarO in which any one of the four conserved cysteines was individually replaced by serine. A promoter assay experiment indicated that the narO gene was usually transcribed, regardless of whether it was cultivated under aerobic or anaerobic conditions. Transcription of the genes encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activated under anaerobic conditions. A putative cis element was identified in the promoter sequence of haloarchaeal denitrifying genes. These results demonstrated a significant effect of NarO, probably due to its oxygen-sensing function, on the transcriptional activation of haloarchaeal denitrifying genes.H. volcanii is an extremely halophilic archaeon capable of anaerobic growth by denitrification. The regulatory mechanism of denitrification has been well understood in bacteria but remains unknown in archaea. In this work, we show that the helix-turn-helix (HTH)-type regulator NarO activates transcription of the denitrifying genes of H. volcanii under anaerobic conditions. A novel cysteine-rich motif, which is critical for transcriptional regulation, is present in NarO. A putative cis element was also identified in the promoter sequence of the haloarchaeal denitrifying genes.
| S-EPMC4800875 | biostudies-other
Project description:In this study, a DeoR/GlpR-type transcription factor was investigated for its potential role as a global regulator of sugar metabolism in haloarchaea, using Haloferax volcanii as a model organism. Common to a number of haloarchaea and Gram-positive bacterial species, the encoding glpR gene was chromosomally linked with genes of sugar metabolism. In H. volcanii, glpR was cotranscribed with the downstream phosphofructokinase (PFK; pfkB) gene, and the transcript levels of this glpR-pfkB operon were 10- to 20-fold higher when cells were grown on fructose or glucose than when they were grown on glycerol alone. GlpR was required for repression on glycerol based on significant increases in the levels of PFK (pfkB) transcript and enzyme activity detected upon deletion of glpR from the genome. Deletion of glpR also resulted in significant increases in both the activity and the transcript (kdgK1) levels of 2-keto-3-deoxy-D-gluconate kinase (KDGK), a key enzyme of haloarchaeal glucose metabolism, when cells were grown on glycerol, compared to the levels obtained for media with glucose. Promoter fusions to a β-galactosidase bgaH reporter revealed that transcription of glpR-pfkB and kdgK1 was modulated by carbon source and GlpR, consistent with quantitative reverse transcription-PCR (qRT-PCR) and enzyme activity assays. The results presented here provide genetic and biochemical evidence that GlpR controls both fructose and glucose metabolic enzymes through transcriptional repression of the glpR-pfkB operon and kdgK1 during growth on glycerol.
| S-EPMC2981212 | biostudies-literature
Project description:Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPR interference (CRISPRi)-mediated repression of the cdrS gene caused slow growth and cell division defects and changed the expression of multiple genes and their products associated with cell division, protein degradation, and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) identified 18 DNA-binding sites of the CdrS protein, including one upstream of the promoter for a cell division gene, ftsZ1, and another upstream of the essential gene dacZ, encoding diadenylate cyclase involved in c-di-AMP signaling, which is implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. IMPORTANCE Cell division is a central mechanism of life and is essential for growth and development. Members of the Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in members of the Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in members of the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here, we identify the small protein CdrS as essential for survival and a central regulator of cell division in the euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways, including synthesis of signaling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.
| S-EPMC8406309 | biostudies-literature
Project description:The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.
| S-EPMC2685553 | biostudies-literature
Project description:Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core alpha1 (psmA), beta (psmB), and alpha2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either alpha1 or alpha2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either beta alone or alpha1 and alpha2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either alpha1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of alpha1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.
| S-EPMC2593203 | biostudies-literature
Project description:Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.
| S-EPMC8316033 | biostudies-literature
Project description:Proteolysis is important not only to cell physiology but also to the successful development of biocatalysts. While a wide-variety of signals are known to trigger protein degradation in bacteria and eukaryotes, these mechanisms are poorly understood in archaea, known for their ability to withstand harsh conditions. Here we present a systematic study in which single C-terminal amino acid residues were added to a reporter protein and shown to influence its levels in an archaeal cell. All 20 amino acid residues were examined for their impact on protein levels, using the reporter protein soluble modified red-shifted GFP (smRS-GFP) expressed in the haloarchaeon Haloferax volcanii as a model system. Addition of hydrophobic residues, including Leu, Cys, Met, Phe, Ala, Tyr, Ile and Val, gave the most pronounced reduction in smRS-GFP levels compared with the addition of either neutral or charged hydrophilic residues. In contrast to the altered protein levels, the C-terminal alterations had no influence on smRS-GFP-specific transcript levels, thus revealing that the effect is post-transcriptional.
| S-EPMC2889427 | biostudies-other
Project description:Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.
| S-EPMC8223754 | biostudies-literature
Project description:The haloarchaeon Haloferax volcanii was shown to contain 145 intergenic and 45 antisense sRNAs. In a comprehensive approach to unravel various biological roles of haloarchaeal sRNAs in vivo, 27 sRNA genes were selected and deletion mutants were generated. The phenotypes of these mutants were compared to that of the parent strain under ten different conditions, i.e. growth on four different carbon sources, growth at three different salt concentrations, and application of four different stress conditions. In addition, cell morphologies in exponential and stationary phase were observed. Furthermore, swarming of 17 mutants was analyzed. 24 of the 27 mutants exhibited a difference from the parent strain under at least one condition, revealing that haloarchaeal sRNAs are involved in metabolic regulation, growth under extreme conditions, regulation of morphology and behavior, and stress adaptation. Notably, 7 deletion mutants showed a gain of function phenotype, which has not yet been described for any other prokaryotic sRNA gene deletion mutant. Comparison of the transcriptomes of one sRNA gene deletion mutant and the parent strain led to the identification of differentially expressed genes. Genes for flagellins and chemotaxis were up-regulated in the mutant, in accordance with its gain of function swarming phenotype. While the deletion mutant analysis underscored that haloarchaeal sRNAs are involved in many biological functions, the degree of conservation is extremely low. Only 3 of the 27 genes are conserved in more than 10 haloarchaeal species. 22 of the 27 genes are confined to H. volcanii, indicating a fast evolution of haloarchaeal sRNA genes.
| S-EPMC3956466 | biostudies-literature