Project description:Transcriptional profiling of Corynebacterium glutamicum cells comparing wild-type cells with cg0196 deletion mutant cells by site-specific gene deletion using the non-replicable integration vector. cg0196 is gene conding transcriptional regulator related carbon metabolism.
Project description:Corynebacterium glutamicum GlxR is a homolog of the cAMP receptor protein. Although over 200 GlxR binding sites in the C. glutamicum genome are predicted in silico, studies on the GlxR physiological function have been hindered by the severe growth defects of a glxR mutant. This study comprehensively identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR binding sites. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a deletion mutant of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased.
Project description:Corynebacterium glutamicum GlxR is a homolog of the cAMP receptor protein. Although over 200 GlxR binding sites in the C. glutamicum genome are predicted in silico, studies on the GlxR physiological function have been hindered by the severe growth defects of a glxR mutant. This study comprehensively identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR binding sites. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a deletion mutant of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. To identify the direct GlxR targets, we immunoprecipitated DNA from a strain expressing a Strep-tag II-tagged GlxR-protein using an anti-Strep-tag II antibody. To investigate effect of depletion of cAMP by deletion of the cyaB gene, which encodes the sole adenylate cyclase in C. glutamicum, on GlxR binding in vivo, we immunoprecipitated DNA from a cyaB deletion strain expressing a Strep-tag II-tagged GlxR-protein using an anti-Strep-tag II antibody. Three or more independent biological replicates were generated in both cases.
Project description:Transcriptional profiling of Corynebacterium glutamicum cells comparing wild-type cells with cg0196 deletion mutant cells by site-specific gene deletion using the non-replicable integration vector. cg0196 is gene conding transcriptional regulator related carbon metabolism. Two-condition experiment, Wild vs. Δcg0196 cells. Independently grown and harvested. One replicate per array.
Project description:To identify genes which are differentially expressed in Corynebacterium glutamicum in the cg2699 deletion strain, we performed DNA microarray analyses of C. glutamicum Δcg2699 compared to the WT.
Project description:To identify genes which are differentially expressed in Corynebacterium glutamicum in the cg2460 deletion strain, we performed DNA microarray analyses of C. glutamicum Δcg2460 compared to the WT.
Project description:The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3′-to-5′ exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596.
Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.