Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens

Abstract

ABSTRACT Clostridium perfringens is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with C. perfringens , a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an Escherichia coli-C. perfringens shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene bgaL ( bgaR -P bgaL ). To measure transcription at the bgaL promoter in pKRAH1, the E. coli reporter gene gusA , encoding β-glucuronidase, was placed downstream of the P bgaL promoter to make plasmid pAH2. When transformed into three strains of C. perfringens , pAH2 exhibited lactose-inducible expression. C. perfringens strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene bglR , encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the yfp - pilB gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in C. perfringens . Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a pilC mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.