Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion.
Colicin V (ColV), an antibacterial peptide toxin, uses a dedicated signal sequence-independent export system for its extracellular secretion in Escherichia coli. The products of at least three genes (a chromosomal tolC gene and two plasmid-born cvaA and cvaB genes) are involved in this process. To characterize the gene products, the cvaA gene was subcloned 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 translational initiation sites. N-terminal peptide sequencing showed that the translation of CvaA starts from a TTG, 11 amino acids upstream of the previously proposed ATG initiation site. CvaA* is translated from an upstream ATG. Expression of both CvaA and CvaA* was induced by the iron chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulated at least partially by Fur. CvaA*-depleted cells were found to secrete less ColV, based on reduced activity in the supernatant, than did wild type, which was recovered by the addition of a plasmid producing CvaA*. Interestingly, CvaA*-depleted and wild-type cells had similar levels of intracellular ColV activity. Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA* depletion. However, CvaA in CvaA*-depleted cells was less stable than that in wild-type cells, indicating that CvaA* may directly or indirectly affect the 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.
Hwang
J
J
Department of Biology, Georgia State University, Atlanta 30303, USA.
Manuvakhova
M
M
Tai
P C
PC
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D018387:Codon, Initiator; D003087:Colicins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D016364:Reading Frames
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D018387:Codon, Initiator; D003087:Colicins; C096447:CvaA protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins