pSE380 vector

There is a high degree of variability in the expression of csgD-dependent, biofilm-forming, and adhesive properties, which is common in Shiga toxin-producing E. coli . Although many O157:H7 serotype strains form little biofilm, conversion to a more robust biofilm phenotype has been observed. In this study, researchers screened different serotype O157:H7 strains for strong Congo red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two main mechanisms conferring a stronger biofilm phenotype were identified: mutations (insertions, deletions, single nucleotide changes) in the rcsB region and stx-prophage excision from the mlrA locus. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties for all variants. Although the RcsB mutant exhibits weaker CR affinity and biofilm properties, it provides additional possibilities for phenotypic presentation through heterologous sequence mutation.

In this study, the rcsB coding region (including the potential ribosome binding site) was amplified using primers RcsB380F-Nco and RcsB380R-Sac and cloned into NcoI-SacI digested pSE380, generating pSE380::rcsB. Complementation plasmids or the pSE380 vector (as a control) were introduced into selected strains with rcsB mutations and their parents by electroporation. Overnight LB cultures were spotted on CRI containing 100 mg L−1 ampicillin and scanned after 48 h of incubation at 25°C, 30°C, and 37°C. In all six red variants tested (with different rcsB mutations or prophage excision), pSE380::rcsB inhibited CR binding in the presence of IPTG at 25°C and 30°C, complementing the suppressive deficiencies of the rcsB mutations or accentuating the wild-type rcsB suppression (in strain 20R2R). RV06 (an extended deletion of the rcsCDB region) also showed repression by pSE380::rcsB, but to a lesser extent, indicating that functional copies of RcsC and RcsD are important for RcsB complementation. Surprisingly, CR affinity in each parental strain was enhanced by pSE380::rcsB under inducing conditions at 37°C, although there was no effect on CR binding under noninducing conditions.

Figure 1. Complement the rcsB mutation using pSE380::rcsB. Parental strains PA20, 380–94, PA7 and selected CR-binding red variants carrying the control plasmid vector pSE380 (–) or the complementation plasmid pSE380::rcsB (+) were spotted on CRI plates containing 100 mg L^-1 ampicillin ± 1mM IPTG at 30°C or 37°C. (Chen C Y, et al. 2016)