期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 77, 期 2, 页码 471-478出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.01536-10
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资金
- USDA/NRICGP [2007-01543]
- NIH [1R21AI088298-01]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R21AI088298] Funding Source: NIH RePORTER
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 beta-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 beta-glucuronidase activity. We mutated gene bglR, encoding a putative beta-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 beta-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.
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