Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of the C. glutamicum deletion mutant of rnc, which encodes an RNase III homolog

ABSTRACT: The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Îrnc 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 Îrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Îrnc and the Îpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3â²-to-5â² 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. Gene expression profile of the wild type at the exponential phase was compared with that of the rnc mutant. Three indepent experiments were performed.

ORGANISM(S): Corynebacterium glutamicum

SUBMITTER: Tomoya Maeda

PROVIDER: E-GEOD-75080 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value â¤ 0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 ÂµM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4Â°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.

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.

Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the M-NM-^TchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum. To identify the influence of ChrSA on global gene expression , DNA microarray analyses were performed with the M-NM-^TchrSA mutant compared to C. glutamicum wild type. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose and either 2.5 M-BM-5M FeSO4 or 2.5 M-BM-5M hemin as iron source. Three biological replicates were performed.

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Transcriptome analysis of the C. glutamicum deletion mutant of rnc, which encodes an RNase III homolog

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