Juliette Morlon

Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate*

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

Variable rate of polypeptide chain elongation for colicins A, E2 and E3.

Abstract Translation in vivo of colicin A messenger RNA was studied. Accumulation of nascent chains of discrete sizes was observed, providing evidence that the rate of chain elongation during colicin A biosynthesis was not uniform. This discontinuous translation was independent of induction of colicin A by mitomycin C and of the bacterial host for colicin A plasmid. A variable rate of chain elongation in vivo was also demonstrated for colicins E2 and E3. The possible roles of codon usage and mRNA secondary structure are discussed.

Lysis protein encoded by plasmid ColA-CA31

SummaryA gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal “signal” region and the mature form is released into the medium together with colicin A.

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

SummaryThe complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×106). 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 andClo 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.

A molecular genetic approach to the functioning of the immunity protein to colicin A.

SummaryA 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.

Physical map of pColA-CA31, an amplifiable plasmid, and location of colicin a structural gene

Evidence showing that the plasmic ColA, derived from strain CA31[pColA] can be amplified in the presence of chloramphenicol is presented. This plasmid has been purified and its Mr-value has been found to be 4.6 X 10(6) or 7 kb. Twelve cleavage sites have been mapped in pColA by using single and double restriction endonuclease digestions. These sites were ordered in relation to the single HindIII site. The other restriction endonucleases used were, respectively, SmaI, AvaI, PstI and HincII. Establishment of the map was helped by hybridization of pColA endonuclease digest products with 32P-labeled colicin A-mRNA. The structural gene for colicin A was contained in a 2.17-kb HincII fragment.

Localization of the structural gene of colicin A on the restriction map of the plasmid pCol A—CA 31 through hybridization with its messenger RNA

Colicinogenic plasmids encode various proteins amongst which colicins and immunity proteins are specific to the plasmid. Colicins are toxins for related species of Escherichia coli and immunity proteins confer protection against these toxins to the plasmid host cell. Like the colicinogenic plasmid Co1 El (pCo1 El), the colicinogenic plasmid Co1 A (PC01 A) is amplifiable in the presence of chloramphenicol [ 11. It thus could be used as a vector allowing production of bank of genes for various organisms [2]. The restriction map of pCo1 A has been made [ 11. Localization of the various genes of this plasmid and in particular that of the structural gene for colicin A (colicin A activity = CM) has been undertaken. Here, we describe the localization of cut gene through hybridization with the colicin A-specific mRNA. The isolation of this mRNA is based upon the fact that --4O mm after induction with mitomycin C, colicin A accounts for nearly 90% of total proteins produced in the strain Citrobacterfreundii CA 3 1 [3].