Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Abstract

BackgroundAlthough useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.Methodology/Principal FindingsAn EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.Conclusions/SignificanceThe new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.

Toxin Plasmids of Clostridium perfringens

“…perfringens carries a chromosomal operon with partial homology to the S. aureus operon encoding components of the Agr quorum-sensing (QS) system. This agr-like operon was shown to regulate CPA and PFO production by C. perfringens type A strain 13, presumably by encoding components of a similar QS system (155,156). It also controls the production of several plasmid-encoded C. perfringens toxins, including CPB2 and CPE expression in type A strain F5603 (157), CPB production in type C strain CN3685 (158) and type B strains CN1793 and CN1795 (159), and ETX production in type D strain CN3718 (154).…”

“…Since the highly conserved agr-like operon present among most or all C. perfringens strains apparently does not encode the AgrA/ AgrC two-component system of the S. aureus Agr QS operon (155,156), it was proposed that C. perfringens uses the VirS/VirR system for responding to Agr-like regulatory system signaling (155). While this putative relationship may yet explain the regulation of some toxins by some C. perfringens strains, recent results indicated that Agr-like regulatory system signaling in this bacterium does not always require the VirS/VirR system (154).…”

Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

“…The banding pattern indicated that each of the mutants had an insertion in a different location and only 2 (ϳ12%) of the clones had multiple insertions (data not shown). These are higher rates of multiple insertions than those reported for the Mu transposition system (0 out of 30 tested [6]) and the EZ-Tn5-based system (0 out of 8 tested [7]), but this rate may be lowered by changing the level of induction of the transposase gene. The chromosomal DNA flanking the site of insertion in 10 of these clones was sequenced (see Materials and Methods); the sites of insertion were randomly distributed around the chromosome, and none of the insertions were in rRNA operons (see Table S1 in the supplemental material).…”

“…A more efficient EZ-Tn5-based random mutagenesis system showed a lower but significant frequency of insertion into rRNA genes (18%) in comparison to the phage Mu-based system (7). Another major limitation of the EZ-Tn5 and phage Mu systems is that both require that the recipient strain be highly transformable by electroporation (6,7).…”

Clostridium perfringenstype C isolates rapidly upregulate their toxin production upon contact with host cells

“…While the LuxS quorum sensing system does not seem to be important for host cell-induced upregulation of toxin production, two studies recently showed that inactivating the agrB gene in C. perfringens type A strain 13 delays the onset of α-toxin and PFO production when this strain grows in bacterial media. 17,18 Those findings open the possibility that the Agr quorum sensing system might also be involved in host-cell induced toxin upregulation by type C isolates.…”