Project description:DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and also occur in select archaea. In the model archaeon Haloferax volcanii, previous work implicated GlpR, a DeoR-type transcriptional regulator, in the transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase (pfkB) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here, we compared transcriptomes of wild-type and ?glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes involved in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under the indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies to function as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that H. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents the broad use of archaea as microbial factories for industrial products. Here, we characterize how sugar uptake and use are regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.

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.

Project description:In this study the transcriptome of Haloferax volcanii DS70 (Wendoloski D, Ferrer C, Dyall-Smith ML. Microbiology. 2001, 147: 959-64), grown under of a variety of conditions, was mapped using a custom genome-wide tiled array consisting of 25 nt probes spaced in 35 nt windows. H. volcanii is a facultative aerobe capable of growth on simple carbon and nitrogen sources, it is amenable to genetic manipulations and has become a widely used model organism for studies on haloarchaeal physiology and global gene regulation. Of particular interest is the identification of genes encoding enzymes needed for growth on simple carbon and nitrogen sources and how these cells respond to limitations in these key nutrients. To address these questions we have compared the RNA populations of cells undergoing balanced growth in completely defined minimal medium (CDM) with succinate-glycerol, pyruvate or lactate as sole carbon sources with RNA from cells grown in complex rich medium (CX) containing yeast extract and tryptone. Data from these studies have identified a core group of genes expressing during balanced growth in either condition as well as specific genes associated with the biosynthesis of amino acids, nucleotides and other key building blocks. The response to nutrient limitation was also investigated. RNA populations from cells entering stationary phase from growth in CDM and CX media were compared to those undergoing balanced growth, and these RNAs were also compared with RNA populations of cells subjected to nitrogen or carbon starvation. Growth in the presence of histidine as the sole source of nitrogen in CDM medium also presented a model for growth with a “poor” nitrogen source. The results of these studies identified specific subpopulations of genes associated with nitrogen limitation and general nutrient limitation associated with the transition to stationary phase. Many of the responsive protein encoding genes were assigned general functions based on their similarity to known proteins; however, in each comparison, approximately half of the affected genes encode proteins classified as unknown or hypothetical proteins. The haloarchaea are also distinct among the Archaea in having multiple genes encoding the general transcription factor proteins TATA binding protein (TBP) and transcription factor IIB (TFB). We observed differential expression of these genes, providing further support for the proposal that alternative TBP-TFB pairings are associated with programmed changes in gene expression in the haloarchaea. RNA was prepared from H. volcanii DS70 cells grown under a variety of conditions. These include: balanced growth in completely defined medium with succinate and glycerol, pyruvate, or lactate as sole carbon source; balanced growth in complex medium with yeast extract and tryptone; starvation for nitrogen or carbon by re-suspending cells, undergoing balanced growth, in media lacking these agents; balanced growth in the presence of low (10% w/v NaCl) and high (20 % or 25% w/v NaCl) salt, and cells entering stationary from growth in CDM or CX media. Four independent cultures were prepared for each growth variable and RNA was isolated from each culture. RNA samples were identified as acceptable for use in array analysis if clear 23S rRNA, 16S rRNA and tRNA-sized bands were present in agarose gel and capillary electrophoresis analysis of the samples. Three RNA samples were chosen for each growth variable and each was used as the source material for a separate chip experiment; these constituted three biological replicates for the growth variable. Data from 48 chip hybridizations, representing 16 growth conditions, were combined for the quantile scaling (Auer H, Newsom DL, Nowak NJ, McHugh KM, Singh S, Yu CY, Yang Y, Wenger GD, Gastier-Foster JM, Kornacker K. BMC Genomics. 2007, 8:111-124) presented in the data analyses. Two additional experiments are included where the RNAs of three independent biological samples were pooled into a single RNA population and used as the source material for an array experiment. These included RNA samples enriched for species less than 500 nt that were isolated from cells undergoing balanced growing, and entry into stationary phase, in CDM or CX media. A second pooled series were obtained from the H. volcanii DS70 mutant strains ASD40 (∆pyrF) and ASD41 (∆pyrF∆hutR) grown in CDM with ammonium ion or histidine as the sole source of nitrogen. Pooled samples are presented as quantile normalized values (Bolstad, B.M., Irizarry, R.A., Astrand, M. and Speed, T.P. Bioinformatics. 2003, 19: 185-193). Total genomic DNA from H. volcanii DS70 was also used as probe, in two independent array experiments, to measure the inherent hybridization to the probes. These data are presented as quantile scaled values (Auer et. al., 2007).

Project description:In this study the transcriptome of Haloferax volcanii DS70 (Wendoloski D, Ferrer C, Dyall-Smith ML. Microbiology. 2001, 147: 959-64), grown under of a variety of conditions, was mapped using a custom genome-wide tiled array consisting of 25 nt probes spaced in 35 nt windows. H. volcanii is a facultative aerobe capable of growth on simple carbon and nitrogen sources, it is amenable to genetic manipulations and has become a widely used model organism for studies on haloarchaeal physiology and global gene regulation. Of particular interest is the identification of genes encoding enzymes needed for growth on simple carbon and nitrogen sources and how these cells respond to limitations in these key nutrients. To address these questions we have compared the RNA populations of cells undergoing balanced growth in completely defined minimal medium (CDM) with succinate-glycerol, pyruvate or lactate as sole carbon sources with RNA from cells grown in complex rich medium (CX) containing yeast extract and tryptone. Data from these studies have identified a core group of genes expressing during balanced growth in either condition as well as specific genes associated with the biosynthesis of amino acids, nucleotides and other key building blocks. The response to nutrient limitation was also investigated. RNA populations from cells entering stationary phase from growth in CDM and CX media were compared to those undergoing balanced growth, and these RNAs were also compared with RNA populations of cells subjected to nitrogen or carbon starvation. Growth in the presence of histidine as the sole source of nitrogen in CDM medium also presented a model for growth with a “poor” nitrogen source. The results of these studies identified specific subpopulations of genes associated with nitrogen limitation and general nutrient limitation associated with the transition to stationary phase. Many of the responsive protein encoding genes were assigned general functions based on their similarity to known proteins; however, in each comparison, approximately half of the affected genes encode proteins classified as unknown or hypothetical proteins. The haloarchaea are also distinct among the Archaea in having multiple genes encoding the general transcription factor proteins TATA binding protein (TBP) and transcription factor IIB (TFB). We observed differential expression of these genes, providing further support for the proposal that alternative TBP-TFB pairings are associated with programmed changes in gene expression in the haloarchaea.

Project description:The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis, and in acetyl coenzyme A (acetyl-CoA) formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase, and pyruvate-ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Furthermore, we show that H. volcanii-during aerobic growth on glucose-excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway and of anaplerosis and report the first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea.IMPORTANCE In this work, we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii The study includes transcriptional analyses, growth experiments with deletion mutants. and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl coenzyme A (acetyl-CoA) and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Furthermore, we analyzed the key players involved in the acetate switch, i.e., in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects of glucose degradation, anaplerosis, and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

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GlpR functions as a global DeoR-type regulator of metabolic gene transcription in the haloarchaeon Haloferax volcanii

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