PCR confirmation of recV gene disruption using ClosTron.
Primers NF1215+NF1356 flanking the ClosTron target site in
recV give a 665 bp product in
630Δerm (WT). After mutagenesis, four
erythromycin resistant colonies were tested and a 2839 bp product was
amplified, indicative of insertion of the group II intron into
recV. These clones were designated
ΔrecV 1-4. B. Diagrammatic
representation of the cwpV DNA switch
orientation-specific PCR assay. Primers NF823+NF826 amplify a
product from OFF, whilst primers NF823+NF825 amplify a product from
ON. C. Analysis of the orientation of the
cwpV DNA switch in C. difficile
clones by orientation PCR. (i), products for the ON and OFF orientations
are amplified from WT. (ii), all four isolated
ΔrecV mutants contain only the OFF orientation
of the cwpV DNA switch, therefore these strains are
referred to as 630ΔrecV OFF. (iii), complementation
of 630ΔrecV OFF using a plasmid encoding
recV (pRecV+) reconstituted the switching
phenotype. (iv), a recVY176F mutant was unable to
complement ΔrecV OFF confirming the key role of
this tyrosine residue in RecV activity. (v),
ΔrecV(pRecV+) was serially sub-cultured
without thiamphenicol selection to enable curing of the pRecV+
plasmid. Four thiamphenicol sensitive colonies were isolated from which
only the ON orientation of the cwpV DNA switch could be
amplified. These strains were therefore designated
ΔrecV ON. D. Colony morphologies
of WT and ΔrecV OFF are similar, however
ΔrecV ON exhibit a smaller, smoother-edged
colony morphology.