The Extracytoplasmic Function-Type Sigma Factor SigM of Corynebacterium glutamicum ATCC 13032 Is Involved in Transcription of Disulfide Stress-Related Genes

Abstract

The gene for the extracytoplasmic function (ECF) sigma factor SigM was deleted from the chromosome of the gram-positive soil bacterium Corynebacterium glutamicum to elucidate the role of the SigM protein in the regulation of gene expression. Comparative DNA microarray hybridizations of the C. glutamicum wild type and sigM-deficient mutant C. glutamicum DN1 revealed 23 genes with enhanced expression in the sigM-proficient strain, encoding functions in the assembly of iron-sulfur clusters (suf operon), thioredoxin reductase (trxB), thioredoxins (trxC, trxB1), chaperones (groES, groEL, clpB), and proteins involved in the heat shock response (hspR, dnaJ, grpE). Deletion of the sigM gene rendered the C. glutamicum cells more sensitive to heat, cold, and the presence of the thiol oxidant diamide. Transcription of the sigM gene increased under different stress conditions, including heat shock, cold shock, and disulfide stress caused by diamide treatment, suggesting a regulatory role for SigM under thiol-oxidative stress conditions. Stress-responsive promoters were determined upstream of the suf operon and of the trxB, trxC, and trxB1 genes. The deduced SigM consensus promoter is characterized by the ؊35 hexamer gGGAAT and the ؊10 hexamer YGTTGR. Transcription of the sigM gene is apparently controlled by the ECF sigma factor SigH, since a sigH mutant was unable to enhance the expression of sigM and the SigM regulon under thioloxidative stress conditions. A typical SigH-responsive promoter was mapped upstream of the sigM gene. The ECF sigma factor SigM is apparently part of a regulatory cascade, and its transcription is controlled by SigH under conditions of thiol-oxidative stress.Corynebacterium glutamicum is a gram-positive, nonsporulating soil bacterium that belongs to the order Actinomycetales, which also includes the genera Mycobacterium and Streptomyces. C. glutamicum is an industrially relevant producer of several L-amino acids (21, 40). Because of its biotechnological importance, the genetic and biochemical background of amino acid biosynthesis has been studied extensively. In recent years, these studies have been broadened by the completion of the genome sequence of the wild-type strain C. glutamicum ATCC 13032 (26, 30). The genome sequence allows the identification of genes by similarity to already known genes in other organisms. However, knowledge about gene expression and its regulation can only be derived from complementary studies. One attempt is based on the detection and classification of transcriptional regulators by bioinformatic methods (6), and the other is based on generating specific regulatory mutants and analyzing changes in gene expression by DNA microarray hybridization and real-time reverse transcription (RT)-PCR assays (25,54,36,7). It is also essential to store this information on transcriptional regulators and their regulatory interactions in the form of specialized databases such as CoryneRegNet, a data warehouse for corynebacterial transcription factors and gene regulatory networks (4). Mo...

Identification of iron-regulated genes of Bifidobacterium breve UCC2003 as a basis for controlled gene expression

“…Following chelation of ferrous iron, B. breve UCC2003 evokes a stress-like response demonstrated by the upregulation of the chaperone GroEL (Bbr_1364). 38,39 Iron-chelation was also shown to upregulate transcription of nrdF (Bbr_1898), encoding a predicted ribonucleotide reductase. The latter enzyme is known in E. coli to catalyze the reduction of ribonucleotides to deoxyribonucleotides as a first step in the pathway for DNA replication, where synthesis of nrd mRNA increases when DNA synthesis is inhibited.…”

RosR (Cg1324), a Hydrogen Peroxide-sensitive MarR-type Transcriptional Regulator of Corynebacterium glutamicum*

“…A transcriptional start site was assumed when at least three of the 10 clones showed the same 5Ј-end and when the other clones contained nonidentical shorter transcripts. The TSS were found to be located 57,196,32, and 45 bp upstream of the proposed translational start sites of cg1150, rosR, cg1848, and cg2329, respectively.…”

Regulation of Quinone Oxidoreductase by the Redox-sensing Transcriptional Regulator QorR in Corynebacterium glutamicum

“…6B). Expression of the trxB1 gene was induced by diamide in the QorRC17S-expressing strain as shown previously by Nakunst et al (15), indicating that cells were subjected to disulfide stress (Fig. 6C).…”

“…4C), but the transcript levels of qor2 and qorR did not respond to H 2 O 2 (data not shown). It has been shown that the sigM transcript level is also increased by diamide but not by H 2 O 2 (15). As C. glutamicum is highly resistant to H 2 O 2 (15), H 2 O 2 is supposed to be unable to induce C. glutamicum oxidative stress response.…”

“…Meanwhile, the species is of increasing interest as a model organism for closely related pathogenic species such as Corynebacterium diphtheriae and Mycobacterium tuberculosis (13,14). In C. glutamicum, the extracytoplasmic function sigma factor SigM is involved in response to disulfide stress, a subcategory of oxidative stress that causes the accumulation of non-native disulfide bonds in the cytoplasm (15). Induction of trxB1 and trxC encoding thioredoxins and trxB encoding thioredoxin reductase by the thiol-specific oxidant diamide is abolished in a sigM disruptant.…”