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:In the analysis of a carbohydrate metabolite pathway, we found that a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase, showed interesting characteristics. After aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced lactate more than 2-fold as compared with the wild-type under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of the pfkB1 deletion and fructose cultivation. On the basis of the metabolome and transcriptome analyses, we identified marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis in this strain. Therefore, the considerably enhanced glucose consumption and organic acid production presumably resulted from a relief of transcriptional repression driven by global regulator SugR owing to the accumulated fructose-1-phosphate. Furthermore, we demonstrated that engineered strains overexpressing the above upregulated genes showed enhanced glucose consumption and organic acid production. The ppc deletion mutant of the engineered strain consumed 1,250 mM glucose and produced 2,390 mM lactate in 48 h under oxygen deprivation, which are 2.6- and 2.7-fold higher than the corresponding parameters of the ppc-deleted wild-type.
Project description:Differential gene expression analysis of C. glutamicum ATCC 13032 in presence of 2.5 mM indole compared to control conditions without indole. C. glutamicum ATCC 13032 cells were cultivated in CGXII minimal medium with 40 g per litre glucose in presence of 2.5 mM indole and harvested during exponential phase (o.d.600 4).
Project description:To identify genes which are differentially expressed in Corynebacterium glutamicum chassis C1 in comparison to the prophage free strain MB001, we performed DNA microarray analyses of C. glutamicum C1 against MB001. 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 5. Four biological replicates were performed.
Project description:Differential gene expression analysis of C. glutamicum C1 in presence of 3 mM indole-alanine dipeptide compared to control conditions without indole-alanine dipeptide. C. glutamicum C1 cells were cultivated in CGXII minimal medium with 40 g per litre glucose in presence or absence of 3 mM indole-alanine dipeptide and harvested during exponential phase (o.d.600 6).
Project description:γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid and widespread in nature from microorganisms to plants and animals. DNA microarray analysis revealed that the transcription of gabTDP was strongly increased in C. glutamicum wild type grown with GABA and urea compared to the same strain cultivated with glucose and urea. Remarkably, the presence of ammonia partially inhibited growth on GABA, and the reasons for it were also investigated in this study.