title: Construction and characterization of a lactose-inducible promoter system for controlled gene expression in Clostridium perfringens.
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.
authors.affiliation: Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
authors.forename: Andrea H
authors.initials: AH
authors.lastname: Hartman
authors.forename: Hualan
authors.initials: H
authors.lastname: Liu
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Melville
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003016:Clostridium perfringens / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007785:Lactose / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D010088:Oxidoreductases; C068255:pilB protein, Bacteria; D005966:Glucuronidase; D007785:Lactose