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Complex regulation of the PEP carboxykinase gene pck and characterization of its GntR-type regulator IolR as a repressor of myo-inositol utilization genes in Corynebacterium glutamicum

ABSTRACT: DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants ΔramA, ΔgntR1ΔgntR2, and ΔiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5’-KGWCHTRACA-3’) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

ORGANISM(S): Corynebacterium glutamicum ATCC 13032

PROVIDER: GSE44812 | GEO | 2013/07/29

SECONDARY ACCESSION(S): PRJNA191907

REPOSITORIES: GEO

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Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants M-NM-^TramA, M-NM-^TgntR1M-NM-^TgntR2, and M-NM-^TiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the M-NM-^TiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5M-bM-^@M-^Y-KGWCHTRACA-3M-bM-^@M-^Y) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. To identify genes that are potentially regulated by IolR, the transcriptome profile of the iolR deletion strain was compared to that of the WT using DNA microarray analysis. The strains were grown in CGXII minimal medium with 4% (wt/vol) glucose as sole carbon source and RNA was isolated from cells harvested in the early exponential growth phase (OD600 of about 5). The transcriptome comparison was performed in triplicate starting from independent cultures. The second replicate included a dye-swap.

Project description:The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An IpsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Analysis of the polar lipid fraction of the cell wall revealed the absence of inositol-derived lipids. The phenotype of the C. glutamicum M-NM-^TipsA mutant was complemented by homologues from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. To identify genes which were regulated by IpsA (Cg2910), we performed DNA microarray analyses of ATCC 13032 M-NM-^TipsA against wild type. For this purpose RNA was isolated from cells cultivated in CGXII minimal medium with 2% glucose (w v-1) and harvested in the exponential growth phase at an OD600 of 1. Three biological replicates were performed.

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Complex regulation of the PEP carboxykinase gene pck and characterization of its GntR-type regulator IolR as a repressor of myo-inositol utilization genes in Corynebacterium glutamicum

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