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Activity and transcriptional regulation of bacterial protein-like glycerol-3-phosphate dehydrogenase of the haloarchaea in Haloferax volcanii.

ABSTRACT: 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.

SUBMITTER: Rawls KS

PROVIDER: S-EPMC3165503 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Activity and transcriptional regulation of bacterial protein-like glycerol-3-phosphate dehydrogenase of the haloarchaea in Haloferax volcanii.

Rawls Katherine S KS Martin Jonathan H JH Maupin-Furlow Julie A JA

Journal of bacteriology 20110701 17

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 neighborh ...[more]

PMID: 21725010

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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: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.

Dataset Information

Activity and transcriptional regulation of bacterial protein-like glycerol-3-phosphate dehydrogenase of the haloarchaea in Haloferax volcanii.

Publications

Activity and transcriptional regulation of bacterial protein-like glycerol-3-phosphate dehydrogenase of the haloarchaea in Haloferax volcanii.

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