GENETIC-ANALYSIS OF THE COLICIN-V SECRETION PATHWAY

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli th rough a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for th e CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replace d with the OmpA signal sequence, the Sec system can localize ColV to t he periplasm. Periplasmic ColV is lethal to cells lacking the ColV imm unity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/T olC mediated secretion. Results indicate that low levels of ColV may b e present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various expo rter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processin g to occur. In contrast, processing occurs normally, albeit more slowl y, in a TolC mutant.