Project description:Previous studies with Corynebacterium diphtheriae and Mycobacterium species revealed that the transcriptional regulator DtxR and its ortholog IdeR play a central role in the control of iron metabolism. In the present work, we used genome-based approaches to determine the DtxR regulon of Corynebacterium glutamicum, a nonpathogenic relative of C. diphtheriae. First, global gene expression of a dtxR deletion mutant was compared with that of the wild type using DNA microarrays. Second, we used a computer-based approach to identify 117 putative DtxR binding sites in the C. glutamicum genome. In the third step, 74 of the corresponding genome regions were amplified by PCR, 51 of which were shifted by the DtxR protein. Finally, we analyzed which of the genes preceded by a functional DtxR binding site showed altered mRNA levels in the transcriptome comparison. Fifty-one genes organized in 27 putative operons displayed an increased mRNA level in the DeltadtxR mutant and thus are presumably repressed by DtxR. The majority of these genes are obviously involved in iron acquisition, three encode transcriptional regulators, e.g., the recently identified repressor of iron proteins RipA, and the others encode proteins of diverse or unknown functions. Thirteen genes showed a decreased mRNA level in the DeltadtxR mutant and thus might be activated by DtxR. This group included the suf operon, whose products are involved in the formation and repair of iron-sulfur clusters, and several genes for transcriptional regulators. Our results clearly establish DtxR as the master regulator of iron-dependent gene expression in C. glutamicum.
Project description:Comparative analysis of Corynebacterium glutamicum transcriptome in response to expression of Egfp under the change of dissolved oxygen in bioreactor
Project description:Optimize SNP genotyping probes and demonstrate a new P. falciparum microarray platform that includes CGH and resequencing probes on the same chip
Project description:The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host's sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by approximately 25% to 4.9 g/L isobutanol in a pycldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.
Project description:We have reported a transcription profile of an adapted Corynebacterium glutamicum that showed enhanced oxidative stress resistance. To construct an artificial oxidative stress-resistant strain, gene clusters in the ?-ketoadipate pathway, which were up-regulated in the adapted strain, were artificially expressed in the wild-type C. glutamicum. The wild-type strain was unable to grow under 2 mM H2O2 containing minimal medium, while the strains expressing pca gene clusters restored growth under the same medium, and the pcaHGBC expression showed the most significant effect among the gene clusters. The expressions of pca gene clusters also enabled the wild-type to increase its resistance against oxidative stressors, such as diamide and cumene hydroperoxide, as well as H2O2. The oxidative stress tolerance of the strain was correlated to the reactive oxygen species (ROS)-scavenging activity of the cell extract. The reason for the enhanced oxidative stress-resistance of C. glutamicum and its applications on the synthetic strain development are discussed.
Project description:DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.
Project description:Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope.Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37?±?1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin.Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.