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.

FIG. 1.

Vector map of plasmid pMTL-SC1. Expression of the hyperactive mariner transposase gene Himar1 C9 was driven by the C. difficile toxin B promoter, PtcdB. The control plasmid pMTL-SC0 was identical, except that there was no promoter driving expression of the transposase gene. The plasmid backbone consisted of the pBP1 replicon of C. botulinum (repA and orf2), the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB, the Gram-negative replicon ColE1, and the conjugal transfer function traJ. The whole mariner element (i.e., transposase gene and catP mini-transposon) can be excised as an SbfI fragment. The transcriptional terminators (Ω) are identical in sequence to those found immediately downstream of the fdx gene of Clostridium pasteurianum and the CD0164 open reading frame of C. difficile 630. This vector conforms to the pMTL80000 modular system for Clostridium shuttle plasmids (8).

FIG. 2.

PCR screens of 17 randomly selected pMTL-SC1-derived Tm^r clones. Genomic DNA prepared from each clone was screened for the transposon-based catP gene (A), the plasmid-based Himar1 C9 transposase gene (B), and an uninterrupted chromosomal tcdB promoter sequence (C). Lane M, 1-kb ladder (Promega); lane P, pMTL-SC1; lane wt, wild-type C. difficile R20291; lanes 1 to 17, pMTL-SC1-derived Tm^r clones 1 to 17.

FIG. 3.

Southern hybridization analysis of pMTL-SC1-derived Tm^r clones. Genomic DNA samples were digested with HindIII. The membrane was probed for the transposon-based catP sequence. Lane wt, wild-type C. difficile R20291; lanes 1 to 17, pMTL-SC1-derived Tm^r clones 1 to 17.

FIG. 4.

Genetic map of mariner transposon insertions. Sixty independent transposon insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for C. difficile R20291 (Refseq number NC_013316; GenBank accession number FN545816) (28).