The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey.
The ccd operon of the F plasmid contributes to the high stability of the episome by postsegregational killing of plasmid-free bacteria. It contains two genes, ccdA and ccdB, which are negatively autoregulated at the level of transcription, probably by a complex comprising the two gene products. Using the bacterial gyrA462 CcdB resistance mutation and a Pccd-lacZ transcriptional fusion, we have obtained evidence that the CcdB protein by itself has no regulatory activity or operator DNA-binding affinity and needs CcdA in order to effect transcriptional control. The ccd killing mechanism is based on the poison-antidote principle. The CcdB protein is cytotoxic, poisoning DNA-gyrase complexes, while CcdA antagonizes this activity. In order to define functional domains of the CcdA antidote involved in the anti-killer effect, autoregulation or both, we introduced several missense or amber mutations into the CcdA protein by directed mutagenesis. We report on missense CcdA proteins that have lost their autoregulatory properties but are still able to antagonize the lethal activity of CcdB. We show that the five carboxy-terminal amino acid residues of the antidote protein are not required for the antidote effect or for autoregulation. Several missense CcdA polypeptides were generated by suppression of nonsense codons. Two substitutions lead to CcdB-promoted killing: glutamine 33-->cysteine and glutamine 33-->phenylalanine.
Salmon
M A
MA
Département de Biologie Moléculaire, Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Van Melderen
L
L
Bernard
P
P
Couturier
M
M
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001433:Bacteriolysis; D001483:Base Sequence; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004249:DNA Damage; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005144:F Factor; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013489:Suppression, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase; D004250:DNA Topoisomerases, Type II