title: Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.
abstract: Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.
authors.affiliation: Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA.
authors.forename: Hualan
authors.initials: H
authors.lastname: Liu
authors.forename: Laurent
authors.initials: L
authors.lastname: Bouillaut
authors.forename: Abraham L
authors.initials: AL
authors.lastname: Sonenshein
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Melville
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics; D003016:Clostridium perfringens / Q000145:classification / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005816:Genetic Complementation Test; D009068:Movement / Q000502:physiology*; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C111652:mariner transposases; D019895:Transposases