GlpR functions as a global DeoR-type regulator of metabolic gene transcription in the haloarchaeon Haloferax volcanii
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ABSTRACT:
INSTRUMENT(S): Roche-NimbleGen-Slide-512049-A02
SUBMITTER: 
Amy K Schmid
PROVIDER: GSE77589 | GEO | 2017-02-01
SECONDARY ACCESSION(S): PRJNA310954
REPOSITORIES: GEO
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Project description:Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.
1000-01-01 | S-EPMC2698487 | BioStudies
Project description:Glycerol is a primary energy source for heterotrophic haloarchaea and a major component of "salty" biodiesel waste. Glycerol is catabolized solely by glycerol kinase (encoded by glpK) to glycerol-3-phosphate (G3P) in Haloferax volcanii. Here we characterized the next critical step of this metabolic pathway: the conversion of G3P to dihydroxyacetone phosphate by G3P dehydrogenase (G3PDH). H. volcanii harbors two putative G3PDH operons: (i) glpA1B1C1, located on the chromosome within the neighborhood of glpK, and (ii) glpA2B2C2, on megaplasmid pHV4. Analysis of knockout strains revealed that glpA1(and not glpA2) is required for growth on glycerol. However, both glpA1 and glpA2 could complement a glpA1 knockout strain (when expressed from a strong promoter in trans) and were required for the total G3PDH activity of cell lysates. The glpA1B1C1, glpK, glpF(encoding a putative glycerol facilitator), and ptsH2(encoding a homolog of the bacterial phosphotransferase system protein Hpr) genes were transcriptionally linked and appeared to be under the control of a strong, G3P-inducible promoter upstream of glpA1. Overall, this study provides fundamental insights into glycerol metabolism in H. volcanii and enhances our understanding of central metabolic pathways of haloarchaea.
1000-01-01 | S-EPMC3165503 | BioStudies
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 ?-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.
2010-01-01 | S-EPMC2981212 | BioStudies
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 ΔglpR 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.
2018-01-01 | S-EPMC6088158 | BioStudies
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).
2010-09-08 | E-GEOD-18066 | ArrayExpress
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. Overall design: 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).
2010-09-08 | GSE18066 | GEO
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.
2014-01-01 | S-EPMC3956466 | BioStudies
Project description:Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general.We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.
2010-01-01 | S-EPMC2841640 | BioStudies
Project description:Extracellular DNA is found in all environments and is a dynamic component of the microbial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA concentrations measured in nature-a potential rich source of carbon, nitrogen, and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration, and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent, and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA. Additionally, fluorescence microscopy showed that labeled DNA co-localized with H. volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in DNA processing at the cell surface, and deletion of Hvo_1477 created a strain deficient in the ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.
2014-01-01 | S-EPMC3929857 | BioStudies
Project description:The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.
2008-01-01 | S-EPMC2223738 | BioStudies