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Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis.

ABSTRACT: Clostridium perfringens is a source of food poisoning in humans and animals because of production of a potent enterotoxin (CPE). To study the regulation of the cpe gene in C. perfringens, we cloned and sequenced the cpe promoter regions and N-terminal domains from three strains. The cpe promoter region from one strain contained a 45-bp insertion compared with previously published sequences. This insertion was also found in two (of five) other Cpe+ strains. cpe gene expression in C. perfringens was measured by using translational fusions of each promoter type to the Escherichia coli gusA gene, which codes for beta-glucuronidase. For either promoter type, cpe-gusA expression was undetectable throughout exponential growth but increased dramatically at the beginning of the stationary phase. To measure cpe expression in Bacillus subtilis, cpe-gusA fusions were integrated into the B. subtilis chromosome. Both types of promoter exhibited moderate expression during exponential growth; cpe expression increased threefold at the beginning of the stationary phase. Transcriptional start sites were determined by primer extension and in vitro transcription assays. For C. perfringens, both types of promoter gave the same 5' end, 197 bp upstream of the translation start (50 bp downstream of the 45-bp insertion). In B. subtilis, however, the 5' end was internal to the 45-bp insertion, suggesting the use of a different promoter than that utilized by C. perfringens.

SUBMITTER: Melville SB

PROVIDER: S-EPMC303301 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis.

Melville S B SB Labbe R R Sonenshein A L AL

Infection and immunity 19941201 12

Clostridium perfringens is a source of food poisoning in humans and animals because of production of a potent enterotoxin (CPE). To study the regulation of the cpe gene in C. perfringens, we cloned and sequenced the cpe promoter regions and N-terminal domains from three strains. The cpe promoter region from one strain contained a 45-bp insertion compared with previously published sequences. This insertion was also found in two (of five) other Cpe+ strains. cpe gene expression in C. perfringens w ...[more]

PMID: 7960138

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Project description:Clostridium perfringens (CP) is the principal pathogenic bacterium of chicken necrotic enteritis (NE), which causes substantial economic losses in poultry worldwide. Although probiotics are known to provide multiple benefits, little is known about the potential effects of Bacillus subtilis (B. subtilis) application in preventing CP-induced necrotic enteritis. In this study, 450 male Arbor Acres broilers were divided into 5 experimental treatments: A: basal diet (control group); B: basal diet and CP challenge (model group); C: CP challenge+10 mg/kg enramycin (positive control group); D: CP challenge+4 × 107 CFU/kg of feed B. subtilis PB6 (PB6 low-dosage group); and E: CP challenge+6 × 107 CFU/kg of feed B. subtilis PB6 (PB6 high-dosage group). There were 6 replicate pens per treatment with 15 broilers per pen. The present research examined the effect of Bacillus subtilis PB6 (B. subtilis PB6) on growth performance, mRNA expression of intestinal cytokines and tight junctions, and gut flora composition in broilers challenged with CP. The entire experiment was divided into two phases: the non-CP challenge phase (d0-18) and the CP challenge phase (d18-26). PB6 did not increase the growth performance during the first stage, but the PB6 high-dosage group was found to have larger body weight gain and ADFI during the CP challenge stage. Feed supplementation with PB6 reduced the lesion score of challenged chicks, with increased tight junction-related gene expression (occludin and ZO-1) and decreased TNF-α expression compared with CP-infected birds. A decrease in the abundance of Clostridium XI, Streptococcus, and Staphylococcus was observed after CP infection (P < 0.05), while supplementation with PB6 restored the ileal microbial composition. In conclusion, administration of B. subtilis PB6 improved growth performance, enhanced intestinal barrier function, and mitigated intestinal inflammation/lesions, which might be due to its restoring effects on the ileal microbial composition in CP-challenged broilers.

Project description:BackgroundB. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength.ResultsHere, site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The transcription level from four mutants was increased. Production of beta-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of beta-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The beta-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated.ConclusionsIn this study, we obtained a mutated promoter Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.

Dataset Information

Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis.

Publications

Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis.

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