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. 2012 Sep 3:13:445.
doi: 10.1186/1471-2164-13-445.

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

Tobias Busche  1 , Radoslav Silar, Martina Pičmanová, Miroslav Pátek, Jörn Kalinowski
Affiliations

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

Tobias Busche et al. BMC Genomics. .

Abstract

Background: The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response.

Results: Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed.

Conclusions: The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.

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Figures

Figure 1
Figure 1
Genetic map of the sigH-rshA operon, its Northern hybridization analysis in C. glutamicum RES167 and its deletion derivatives. A. Genetic map of the sigH-rshA region showing locations and sizes of deletions in the chromosomes of strains C. glutamicum ΔrshA, DN2 and AS1, predicted sizes of respective sigH-rshA and rshA transcripts (arrows) and locations of probes used for Northern hybridizations. Promoters are indicated with bent arrows and the terminator with a hairpin symbol. B. Northern blot using a sigH probe (left panel) and an rshA probe (right panel) hybridized with total RNA extracted from: RES167 cells (lane 1); DN2 cells (ΔsigH deletion; lane 2); AS1 cells (ΔsigHrshA deletion; lane 3). The estimated lenghts of the detected transcripts (left) and their designations (right) are indicated. The sizes of the fragments in the RNA marker are indicated with arrows.
Figure 2
Figure 2
Determination of transcriptional start points of the sigH and rshA genes and sequences of their promoter regions. (a) and (b) Determination of the sigH transcription start sites (TSP) by primer extension analysis. The bottom peaks (PEX) represent cDNA synthesized in the reverse transcription using RNA from C. glutamicum (pET2sigH) and C. glutamicum (pET2sigH4), respectively. The smaller peaks were not reproducibly observed in the repeated experiments. The peaks (A, C, G, T) represent the products of sequencing reactions carried out with the same fluorescent-labeled primer as that used for reverse transcription. (c) Nucleotide sequence of the sigH upstream region. TSPs and the proposed −35 and −10 promoter elements are in bold and underlined. Transcription initiation is indicated by the bent arrows. The proposed binding site for the LexA regulator is boxed and the initiation codons (in bold) of the genes sigH and cg0875 are indicated with hollow arrows. (d) Determination of rshA TSP. (e) Nucleotide sequence of the rshA upstream region. The stop codon (in bold) of sigH is indicated with the black dot. Note that the sequences (c) and (e) are complementary and reversed to those deduced from the peaks generated by the sequencer.
Figure 3
Figure 3
Microarray analysis of the C. glutamicum RES167 strain compared with its Δ rshA mutant DN2. Ratio/intensity plot obtained from the DNA microarray comparing the transcriptomes of RES167 and DN2 is shown. Total RNA was isolated from two biological replicates grown in minimal CGXII medium to the exponential phase and used for hybridization. Genes with increased amounts of mRNA in the ΔrshA strain have positive ratios, while genes with a higher mRNA amount in the RES167 strain have negative ratios, indicated with green diamonds (upregulated) or red triangles (downregulated) respectively; those not exhibiting differential expression are indicated with grey spots. M values of higher than +0.6 or lower than −0.6 (corresponding to fold changes of 1.52 and 0.66, respectively) were considered to be significant. The relevant genes are indicated by their names or desigations from the C. glutamicum genome sequence (GenBank NC_006958), underlined genes were previously described as SigH-dependent.
Figure 4
Figure 4
Relative transcript levels of selected potential SigH-dependent genes in C. glutamicum Δ rshA/C. glutamicum RES167 measured by q-RT-PCR. The data obtained for the RES167 strain served as a reference and the respective values were set to 1.0 on the logarithmic scale. Three biological replicates for the ΔrshA strain and four replicates for the RES167 strain were analysed in duplicate. SD values are shown as error bars.
Figure 5
Figure 5
Determination of transcriptional start points of uvrA and dnaJ2 genes. (a) Determination of uvrA TSP. A portion of the nucleotide sequence derived from the sequencing peaks is shown below, TSP is in bold and underlined. (b)uvrA promoter sequence. TSP (+1) and the proposed −35 and−10 promoter elements are in bold and underlined. (c) Determination of dnaJ2 TSP. (d)dnaJ2 promoter sequence. Note that the sequences at (b) and (d) are reversed and complementary to those shown in (a) and (c).
Figure 6
Figure 6
Sequences of presumed C. glutamicum SigH-dependent promoters. Putative −10 and −35 regions (a spacer of 16 -19 nucleotides) and TSPs (+1) are highlighted in bold. Dashes indicate gaps introduced to align the −35 element. Positions in the C. glutamicum consensus with a single nucleotide occurrence of over 80% are in bold letters, K= G or T; Y= C or T; R= A or G;W = A or T. The sequence reported by Halgasova et al.[22] is from C. glutamicum CCM251, the sequences reported by Ehira et al.[14] are from C. glutamicum R, and the others are from C. glutamicum ATCC 13032.
Figure 7
Figure 7
Distribution of nucleotides within the −35 and −10 core regions of C. glutamicum SigH-dependent promoters. The percentage occurrence of a nucleotide at a particular position is represented by the size of the nucleotide symbol (A, C, G, T) using Weblogo [57]. This analysis is based on 45 presumed SigH-dependent promoters.
Figure 8
Figure 8
Extended model of the SigH regulatory network in C. glutamicum. Conditions that deplete thiols by oxidation or alkylation cause the oxidation of cysteine residues inside RsrA. RsrAox is released from SigH, which then binds to the RNA polymerase (RNAP) and initiates the transcription of its regulon. Direct induction of trxBC/B1, mca, mshC and mtr genes that are involved in the disulphide response generate and recycle/reduce the thiols thioredoxin (Trx) and Mycothiol (MSH) to reverse the oxidation of RsrA, restore thiol redox balance and re-establish the binding of RshA to SigH. The direct induction of rshA as a single transcript amplifies the shutdown of the SigH-dependent response after the cells have coped with the stress. The SigH regulon includes part of the SOS response and the heat-shock regulon, including the HspR and ClgR regulatory networks, which are responsible for protein quality control.
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