title: Construction and analysis of chromosomal Clostridium difficile mutants.
abstract: Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.
authors.affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Vic. 3800, Australia.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: O'Connor
authors.forename: Dena
authors.initials: D
authors.lastname: Lyras
authors.forename: Kylie A
authors.initials: KA
authors.lastname: Farrow
authors.forename: Vicki
authors.initials: V
authors.lastname: Adams
authors.forename: David R
authors.initials: DR
authors.lastname: Powell
authors.forename: Jason
authors.initials: J
authors.lastname: Hinds
authors.forename: Jackie K
authors.initials: JK
authors.lastname: Cheung
authors.forename: Julian I
authors.initials: JI
authors.lastname: Rood
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D009154:Mutation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins