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 thiol-oxidative 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.

FIG. 1.

Comparative analysis of sigma factor proteins and sigM gene regions of actinobacterial species. (A) Phylogenetic tree of actinobacterial sigma factors including the seven sigma factors of C. glutamicum ATCC 13032. The unrooted phylogenetic tree for different actinobacterial sigma factors is shown. Abbreviations: Cg, C. glutamicum; Ce, C. efficiens; Cd, C. diphtheriae; Cj; C. jeikeium; Ma, M. avium; Mt, M. tuberculosis; Sc, S. coelicolor. Scale bar, 0.1% amino acid substitution. (B) Comparison of the sigM gene regions of different actinobacteria. For corynebacteria, mycobacteria, nocardia, and streptomycetes, the organization of the sigM gene region is shown. The locations of the genes sigM, trxB, trxC, and cwlM are indicated. In mycobacteria, streptomycetes, and nocardia, a further gene of unknown function is located downstream of sigM.

FIG. 2.

Ratio-versus-intensity plot of the DNA microarray hybridization comparing the gene expression of C. glutamicum parental strain RES167 with that of sigM mutant C. glutamicum DN1. Genes were regarded as differentially expressed with m values of ≥1 or ≤−1. The m value is the log₂ normalized expression ratio. Genes with enhanced expression in C. glutamicum RES167 are marked by black diamonds; genes with decreased expression are shown as black triangles. Differentially expressed genes of C. glutamicum RES167 are labeled by names or identifiers; genes not differentially expressed are shown as gray dots.

FIG. 3.

CFU of C. glutamicum parental strain RES167 and sigM mutant C. glutamicum DN1 after the application of different stress conditions. Cultures of C. glutamicum RES167 and DN1 were grown in minimal medium (MM1), and cells were harvested during exponential growth at an optical density of 7. The following stress conditions were applied for 15 min: heat shock at 50°C, cold shock at 10°C, disulfide stress caused by 2 or 10 mM diamide, oxidative stress caused by 1% hydrogen peroxide, salt stress caused by 1 M NaCl, and alcohol stress caused by 10% ethanol. Following stress treatment, the colony-forming ability of the cultures was determined with three biological and two technical replicates. CFU percentages are represented by black (RES167) and gray (DN1) columns.

FIG. 4.

Relative mRNA levels of the sigM gene of a C. glutamicum culture following the application of different stress conditions. The stress conditions were heat shock at 50°C, cold shock at 10°C, disulfide stress caused by 2 mM diamide, oxidative stress caused by 1% hydrogen peroxide, salt stress caused by 1 M NaCl, and alcohol stress caused by 10% ethanol. After stress application, the cells were washed with MM1 medium and total RNA was extracted. The relative amounts of sigM transcripts were subsequently determined by real-time RT-PCR and calculated in relation to that of an untreated control culture. The measurements were carried out with three biological and two technical replicates.

FIG. 5.

Classification of SigM-dependent genes differing in their responses to disulfide stress. The genetic organization of the respective gene regions is shown. Stem-loops denote rho-independent transcriptional terminators (13). Black boxes indicate experimentally mapped promoters, and white boxes indicate promoters that where found by sequence similarity. RT-PCR ratios were deduced from comparisons of gene expression in C. glutamicum RES167 and sigM mutant C. glutamicum DN1 (first number above the genes), as well as from comparison of C. glutamicum RES167 treated with 2 mM diamide and a C. glutamicum RES167 control (second number above the genes). n.s., no significant change in gene expression.

FIG. 6.

Promoter sequences of disulfide stress-responsive genes. (A) Alignment of SigM-dependent promoter regions of differentially expressed genes of C. glutamicum RES167. The transcriptional start sites (+1) were determined by RACE-PCR, with total RNA from a C. glutamicum RES167 culture treated with diamide. sigR (Sc), consensus sequence of SigR-dependent promoters from S. coelicolor (50). (B) Promoter sequences of other C. glutamicum genes potentially transcribed with the help of SigM. The promoter regions (−10 and −35) were detected by similarities to the consensus sequence shown in panel A. (C) Nucleotide sequence of the suf operon promoter region of C. glutamicum. The transcriptional start sites (+1) and deduced promoter elements (−10 and −35) of the housekeeping promoter (underlined) and the stress-responsive SigM promoter (overlined) were determined by RACE-PCR. (D) Mapped promoter of the sigM gene of C. glutamicum. sigH (Mt), consensus sequence of SigH-dependent promoters from M. tuberculosis (33). Abbreviations: R, A or G; Y, C or T.

FIG. 7.

Relative mRNA levels measured by RT-PCR of differentially expressed genes upon disulfide stress caused by diamide (2 mM) in C. glutamicum RES167 (WT [wild type]), the sigH-deficient mutant C. glutamicum DN2, and the sigM-deficient mutant C. glutamicum DN1. The mRNA level of an untreated C. glutamicum RES167 culture was used as a reference and set to 1. A, diamide-treated C. glutamicum parental strain RES167; B, untreated sigH-deficient mutant DN2; C, diamide-treated sigH-deficient mutant DN2; D, untreated sigM-deficient mutant DN1; E, diamide-treated sigM-deficient mutant DN1.

FIG. 8.

The regulatory network of the C. glutamicum disulfide stress response. The sigma factor SigH is bound to its anti-sigma factor RshA until the anti-sigma factor senses an oxidative stress by being oxidized and releases the sigma factor. SigH binds to the RNA polymerase and initiates the transcription of the whcE and sigM genes. SigM binds to the RNA polymerase, interacts with WhcE, and initiates the transcription of the SigM-dependent disulfide stress-responsive genes, including thioredoxin-encoding genes. Thioredoxin is able to reduce oxidized groups in RshA, and therefore RshA is again functional and binds SigH.