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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.

The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48 X 10(6]. Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 and Clo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.

ColA is a colicinogenic plasmid of 6.72 kb. It is compatible with ColE1 but not with ColK. Transposon insertion mutagenesis as well as complementation studies have been carried out to investigate the location of the various functional regions of this plasmid. Four independent ColA::Tn1 and one ColA::Tn3 plasmids were isolated and the locations of insertions were determined. From these plasmids, six different deletion mutants were constructed. In addition, various restriction fragments of ColA have been cloned into pUC8 to carry out complementation studies. We have thus confirmed the location of the DNA regions involved in colicin production, colicin release and immunity function. The DNA region involved in conjugal mobility promoted by R64 drd11 has been identified and we have demonstrated that the ColE1 mobility proteins can act in trans on the bom (basis of mobility) site of ColA. The location of this site, as well as the region involved in stable maintenance of ColA, have also been determined. These results are discussed with regard to the homology in nucleotide sequence between ColA and ColE1.