Characterization of a Toxin-Deficient Clostridium perfringens Strain,KZ1340

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an α, θ-, and κ-toxin non-producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (θ-toxin) gene, but the plc (α-toxin) and colA (κ-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin-deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.     

A minimal base‐pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq‐binding small RNA that is induced under glucose‐phosphate stress to cause translational repression and RNase E‐dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31‐nt‐long stretch in the 3′ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre‐annealed with ptsG mRNA by a high‐temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base‐pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base‐pairing region is sufficient to inhibit ptsG translation in the context of full‐length SgrS in vivo. We conclude that SgrS 168–181 is a minimal base‐pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high‐temperature pre‐treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.     

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.