Construction and characterization of a lactose-inducible promoter system for controlled gene expression in Clostridium perfringens

Appl Environ Microbiol. 2011 Jan;77(2):471-8. doi: 10.1128/AEM.01536-10. Epub 2010 Nov 19.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Artificial Gene Fusion
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Clostridium perfringens / genetics*
  • Clostridium perfringens / metabolism
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial*
  • Genes, Reporter
  • Genetic Engineering / methods*
  • Genetic Vectors
  • Genetics, Microbial / methods*
  • Glucuronidase / genetics
  • Glucuronidase / metabolism
  • Lactose / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Molecular Biology / methods*
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism
  • Plasmids
  • Promoter Regions, Genetic*

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • yellow fluorescent protein, Bacteria
  • Oxidoreductases
  • pilB protein, Bacteria
  • Glucuronidase
  • Lactose