Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene ( cpe ) of Clostridium perfringens

Abstract

Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringensenterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpepromoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression ofcpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for β-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpepromoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.

Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A

“…The mechanism of Spo0Aregulated CPE synthesis remains unknown. However, two hypotheses can be envisioned: (i) Spo0A may activate transcription of the cpe gene via activating sporulation-specific sigma factors encoding genes, sigE and sigK, and/or (ii) Spo0A directly activates cpe by binding to the putative 0A box (TGTAGAA) located in the promoter region of the cpe gene [9,10]. Further studies of Spo0A and cpe promoter binding, and sigE and sigK knock-out mutants, should help in understanding the mechanism of Spo0A-regulated CPE synthesis.…”

“…Clostridium perfringens wild-type SM101, spo0A mutant IH101 and the complemented IH101(pMRS123) strains were grown in Duncan-Strong (DS) medium [7] at 37°C for 6 h. These cultures were used to isolate total RNA as previously described [8,10]. The primers CPP68 (5 0 -CAGGAATTGCAAAGGATGGATTGGAAGC-3 0 ) and CPP69 (5 0 -GGCATCTATTTGTCCTCTTCCCC-AAG-3 0 ), which amplified a 619-bp internal spo0A fragment, were used to detect spo0A-specific mRNA in total RNA preparations by RT-PCR analysis with the commercially available Access RT-PCR kit (Promega).…”

“…Substantial experimental and epidemiological evidence [1,5] now indicates that most, if not all, GI symptoms of these C. perfringens associated diseases are caused by the C. perfringens enterotoxin (CPE). Although several studies [6][7][8][9][10] indicated that CPE synthesis and release is associated with sporulation, this association has never been confirmed by gene knock-out studies. Furthermore, the molecular mechanism of sporulation and its role in CPE synthesis and release has not been studied in great detail at the molecular level.…”

C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA

“…In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein [18,21]. In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A [64] and also the production of enterotoxin in this organism seems to be (indirectly) dependent on sporulation [65,66].…”

Erythrophore cell response to food‐associated pathogenic bacteria: implications for detection

“…Salmonella enteritidis ATCC 4931 was cultured in LB overnight at 37°C. Clostridium perfringens SM101 (Zhao and Melville, 1998) was generously provided by Dr Mahfuzur R. Sarker. Briefly, a 0.1 ml aliquot of a frozen stock of C. perfringens SM101 was transferred into 6 ml of fluid thioglycollate (FTG) and then heat shocked for 20 min at 70°C.…”