Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

Abstract

The NfrA protein, an oxidoreductase from the soil bacterium Bacillus subtilis, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the nfrA gene belongs to the class III heat shock genes in B. subtilis. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that nfrA and the gene downstream of nfrA are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding nfrA and ywcH. The nfrA-ywcH operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.The continuous demand of Bacillus subtilis to adapt to everchanging conditions in its natural environment has forced the generation of complex regulatory mechanisms governing the transcription of stress-specific proteins. Stress-inducible genes from B. subtilis in general are subdivided into three groups (17,18). Class I genes are specifically induced by heat stress (17). The well-known chaperonins GroEL, GroES, DnaK, DnaJ, and GrpE are encoded by genes belonging to this group (30,41,48,54,55). The transcription of the respective genes is regulated by HrcA, a transcription repressor which binds to the CIRCE element (43,56,57,59). Genes transcribed in a Bdependent manner constitute class II stress-responsive genes (17, 18). B activity is triggered by different kinds of stress and by starvation (5, 7-9). Members of the last group of stressinduced genes, class III, are induced not by starvation but by several different stressful conditions. The transcription of class III genes is neither repressed by HrcA nor solely dependent on B . The regulator of the clpC operon, which encodes class III proteins, is known (11,27). This operon is transcribed by the activity of RNA polymerases containing B and A (28). Nevertheless, transcription is not induced at the onset of the stationary phase (28), likely because of the activity of this regulator (11,27).Some of the stress-responsive proteins are regulated by two transcription factors. clpC, dps, trxA, opuE, and clpP (1,4,15,29,40,46) are transcribed by RNA polymerase containing either A or B . csbB is under the additional control of X (22). The csb40 operon (50) and the yvyD gene (13) are transcribed from B and H promoters, respectively. This genetic organization enables the bacterial cell to modulate the regulation of the respective genes in response to additional challenges.In this communication, we describe the transcriptional regulation of the nfrA-ywcH operon encoding an oxidoreductase (34, 58) and a p...

Staphylococcus aureus NfrA (SA0367) Is a Flavin Mononucleotide-Dependent NADPH Oxidase Involved in Oxidative Stress Response

“…RNA was isolated from the stressed and unstressed bacteria and was subjected to Northern blot analyses and RT-PCR. In contrast to nfrA expression in B. subtilis (24), no induction of nfrA was observed in response to heat in S. aureus (Fig. 4).…”

“…Regulation of nfrA expression by use of different sigma factors in B. subtilis, however, is still being investigated since Moch et al have identified putative SigA-, SigB-, and SigD-dependent promoter sequences in front of the nfrA gene. Moreover, heat induction of nfrA was still observed in a SigB-and SigD-deficient strain and also in a CtsR-and HrcA-deficient strain (24).…”

“…Effects of different stress stimuli on the expression of nfrA. It was previously determined that expression of the nfrA gene of B. subtilis was induced upon heat shock (24). In a DNA microarray experiment with B. subtilis it was recently shown that nfrA was also induced upon oxidative stress (28).…”

“…NfrA is widely distributed among bacterial species. For example, homologous counterparts were found by (24). The protein homologous to NfrA in E. coli, NfsA, shares 37% amino acids with the NfrA protein of S. aureus.…”

“…Furthermore, primer extension assays revealed that transcription of nfrA starts at promoter sequences typical for SigA-dependent transcription. The nfrA gene of B. subtilis is induced by heat stress (24), as well as by oxidative stress caused by hydrogen peroxide and paraquat (28). Regulation of nfrA expression by use of different sigma factors in B. subtilis, however, is still being investigated since Moch et al have identified putative SigA-, SigB-, and SigD-dependent promoter sequences in front of the nfrA gene.…”