title: A mariner-based transposon system for in vivo random mutagenesis of Clostridium difficile.
abstract: Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (+/-0.4) x 10(-4) per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.
authors.affiliation: Centre for Biomolecular Sciences, School of Molecular Medical Sciences, Nottingham Digestive Diseases Centre, NIHR Biomedical Research Unit, The University of Nottingham, University Park, Nottingham NG72RD, United Kingdom.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Cartman
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D016360:Clostridioides difficile / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology; D015723:Gene Library; D005798:Genes, Bacterial; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D019895:Transposases / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C111652:mariner transposases; D019895:Transposases