Figure 5.

Periplasmic production of CTXΦ pIII-N1 domain or variants in V. cholerae and phenotypic characterization. Precultures of V. cholerae O395 cells carrying various pIN constructs were grown in LB supplemented with MgCl₂ to promote OM integrity and cell growth. A, CTX transduction assay was conducted in triplicate, and CFU were counted on LB or LB supplemented with Cm. Frequency of infection is calculated by dividing the number of transductants (CmR colonies) by the number of O395 recipients. The mean and the standard deviation of the triplicate is presented. For each construct, infection efficiency is expressed as the percentage of infection compared with the receiver strain carrying the empty vector. B, membrane integrity assay. 4 μl of 10-fold dilution of normalized cultures (initial A₅₉₅ = 1) were spotted on LB+amp plates alone or supplemented with 1% DOC. C, percentage of survival to SDS 0.125% of the different strains compared with V. cholerae WT strain carrying a pIN empty vector. For each strain, percentage of growth is calculated as A_{600 nm} of each stain grown in LB+SDS × 100/A_{600 nm} of the WT strain carrying an empty vector and grown in LB. The experiments were conducted in triplicate, and the standard deviation is presented.