CHARACTERIZATION OF IN-FRAME PROTEINS ENCODED BY CVAA, AN ESSENTIAL GENE IN THE COLICIN-V SECRETION SYSTEM - CVAA-ASTERISK STABILIZES CVAA TO ENHANCE SECRETION

Colicin V (CoIV), an antibacterial peptide toxin, uses a dedicated sig nal sequence-independent export system for its extracellular secretion in Escherichia coli, The products of at least three genes (a chromoso mal tolC gene and two plasmid-born cvaA and cvaB genes) are involved i n this process, To characterize the gene products, the cvaA gene was s ubcloned and expressed under the control of T7 RNA polymerase promoter , Two in-frame proteins, CvaA and CvaA, were expressed and identified , DNA sequences predicted that both proteins have two potential transl ational initiation sites, N-terminal peptide sequencing showed that th e translation of CvaA starts from a TTG, 11 amino acids upstream of th e previously proposed ATG initiation site, CvaA is translated from an upstream ATG, Expression of both CvaA and CvaA was induced by the ir on chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulat ed at least partially by Fur, CvaA-depleted cells were found to secre te less ColV, based on reduced activity in the supernatant, than did w ild type, which was recovered by the addition of a plasmid producing C vaA. Interestingly, CvaA*-depleted and wild-type cells had similar le vels of intracellular ColV activity, Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA depletion, H owever, CvaA in CvaA-depleted cells was less stable than that in wild -type cells, indicating that CvaA may directly or indirectly affect t he stability of CvaA, We conclude that CvaA is not essential for ColV secretion but that it enhances the ColV secretion by stabilizing the CvaA protein.