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Analysis of the transcriptional regulator GlpR, promoter elements, and posttranscriptional processing involved in fructose-induced activation of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Haloferax mediterranei.

ABSTRACT: Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5' untranslated region (UTR) were observed, and promoter or 5' UTR swap experiments indicated that the shorter 5' UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5' UTR was significantly higher and the ratio of T8 (with the shorter 5' UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose.

SUBMITTER: Cai L

PROVIDER: S-EPMC3911062 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Analysis of the transcriptional regulator GlpR, promoter elements, and posttranscriptional processing involved in fructose-induced activation of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Haloferax mediterranei.

Cai Lei L Cai Shuangfeng S Zhao Dahe D Wu Jinhua J Wang Lei L Liu Xiaoqing X Li Ming M Hou Jing J Zhou Jian J Liu Jingfang J Han Jing J Xiang Hua H

Applied and environmental microbiology 20131213 4

Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, micr ...[more]

PMID: 24334671

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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

Analysis of the transcriptional regulator GlpR, promoter elements, and posttranscriptional processing involved in fructose-induced activation of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Haloferax mediterranei.

Publications

Analysis of the transcriptional regulator GlpR, promoter elements, and posttranscriptional processing involved in fructose-induced activation of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Haloferax mediterranei.

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