Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid (original) (raw)
Related papers
High-level expression of the colicin A lysis protein
MGG Molecular & General Genetics, 1989
Two plasmids that overproduce the colicin A lysis protein, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed the cal gene near a truncated caa gene in such a way that both gene products were synthesized at high levels following induction. Plasmid CK4 was constructed by insertion of the cal gene downstream from the tac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 ceils. The kinetics of Cal synthesis were examined with [3ss] methionine and [2-3H] glycerol in lpp or lpp § host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Caloverproducing cells, the rate of quasi-lysis was increased but not its extent. In pldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in the same range as that in wildtype cells.
Cloning and expression of the activity and immunity genes of colicins B and M on ColBM plasmids
MGG Molecular & General Genetics, 1984
The activity and immunity genes for colicins B and M on two conjugative ColBM plasmids, pCl139 and pF166, were cloned into pBR322 and pACYC184, respectively. The colicin regions on both recombinant plasmids were identical with regard to restriction endonuclease sites and the arrangement of the genes. They map close to each other in the order cmi cma cbi cba, where cmi denotes the locus that determines immunity to colicin M, ema the structural gene for colicin M, ebi immunity to colicin B, and cba the structural gene for colicin B. With the use of mutants obtained by insertion of the transposon Tn5, and by translation in minicells, the transcriptional polarity of ema and cba was found to be from right to left. ema and eba code for polypeptides with molecular weights of 27,000 and 58,000, respectively. No evidence of biosynthetic precursors was obtained.
Sequence, expression and localization of the immunity protein for colicin B
MGG Molecular & General Genetics, 1988
Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.
Construction and analysis of miniplasmids of the colicin Ib plasmid
Plasmid, 1983
Miniplasmids of the colicin Ib (ColIb) plasmid have been isolated from two TnS-induced mutants of ColIb and their structure determined. These have then been used to order the sequence of restriction endonuclease fragments of the whole plasmid. In addition, the sites of the colicin, colicin immunity, and abortive infection gene have been determined in relation to the restriction sites. By comparison of the miniplasmids with other "I" incompatibility group plsmids, the probable location of the incompatibility gene and the origin of replication have been confirmed.
A molecular genetic approach to the functioning of the immunity protein to colicin A
MGG Molecular & General Genetics, 1986
A plasmid (pColAFI), 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.
Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D
Molecular Microbiology, 1989
The nucleotide sequence of a 2.4kb Dral-£coRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene {cda) and the colicin D immunity gene {cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74683D and that the immunity protein had a molecular weight of 10057D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.
European Journal of Biochemistry, 1984
The nucleotide sequence of the structural gene for the immunity protein to colicin A (cai) has been established. This sequence consists of 534 base pairs. According to the predicted amino acid sequence, the polypeptide chain of this immunity protein comprises 178 amino acids and has a relative molecular mass of 20462. As expected from its localization in the inner membrane, large hydrophobic fragments are found along the polypeptide chain that also contains clusters of mostly positively charged residues. The cai like the ceiA genes encode proteins that are weakly expressed as compared to the corresponding colicins (A and El). Codon usage reflects this difference. In contrast, the four genes for immunity to cloacin DFI 3 and to colicin E3 and for these bacteriocins, all of which are highly expressed and are organized in operon, display similar codon usage. These results are discussed with regards to the possible relationship between expressivity and codon usage.