A molecular genetic approach to the functioning of the immunity protein to colicin A |
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Authors: | Vincent Geli Daniel Baty Veronica Crozel Juliette Morlon Roland Lloubes Franc Pattus and Claude Lazdunski |
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Institution: | (1) Centre de Biochimie et de Biologie Moléculaire du C.N.R.S., 31 Chemin Joseph Aiguier, BP 71, F-13402 Marseille Cedex 9, France |
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Abstract: | Summary A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH2-and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124–143 abolish the protecting function of Cai. |
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Keywords: | Colicins Immunity protein Membrane-spanning protein Site-directed mutagenesis |
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