The promoters of the genes for colicin production, release and immunity in the ColA plasmid: effects of convergent transcription and Lex A protein (original) (raw)
Related papers
Characterization of thecea gene of the CoIE7 plasmid
MGG Molecular & General Genetics, 1992
The complete nucleotide sequence (1731 nucleotides) of the gene encoding colicin E7 (cea) of plasmid ColE7-K317 was determined. This sequence encoded a deduced polypeptide of 576 amino acids of molecular weight 61349 Da. Comparison of the nucleotide and amino acid sequences of cea E7 with those of other E-group colicins revealed that colicin E7 was closely related to colicin E2, both in gene sequence and in predicted secondary structure of the deduced protein. Judging from the results of cross-immunity tests, we postulated that ColE7 is probably a proximate ancestor of ColE2 and ColE8. Based on results from colicin production tests on cells harboring a 5' end deleted form of the cea E7 gene, we propose, that a previously unknown, non-inducible promoter may be involved in regulation of the constitutive expression of the cea E7 gene.
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 ยง 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.
Nucleic Acids Research
We have constructed two new promoter probe vectors which carry a polylinker derived from plasmid pUC19 proximal to the 5' end of a promoter-less galactokinase gene. Using these two vectors we have demonstrated that the ColE3imm gene and the ColE8imm gene present on the ColE3-CA38 plasmid have their own promoters, independent of the SOS promoter of the colicin E3 structural gene. The activity of two terminators, one located proximal to the 5' end of the ColE8imm gene, the other located proximal to the 5' end of the lys gene, were shown by a comparison of the galactokinase activity conferred by several of the recombinant plasmids.
Microbiology, 1984
Escherichia coli W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins. By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the EcoRI site (4.0 kb on the restriction map) and the PuuII site (3.68 kb) of the ColE3-CA38 plasmid. This placed the colicin E8 immunity gene between the colicin E3 immunity gene and Zys, the region which determined mitomycin C sensitivity. Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes. In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity. The phenotype conferred by plasmids with a transposon inserted into the lys region of ColE3-CA38 was dependent upon the site of insertion.
Methods in Molecular Biology - E. coli Plasmid Vectors
ColE1 uses an extensive RNA primer for leading-strand synthesis. The RNAII preprimer is transcribed from the RNAII promoter by host RNA polymerase. RNAII forms a persistent RNA-DNA hybrid at the plasmid origin of replication. This hybrid is cleaved by RNase H and the resulting free 3'OH group on the cleaved RNAII acts as a primer for continuous leading-strand synthesis, catalyzed by host DNA polymerase I. ColE1 regulates its copy number with a short RNA countertranscript, RNAI. This species is expressed constitutively from the strong RNAI promoter, is nontranslated, and is fully complementary to part of RNAII. The interaction of RNAI with RNAII results in an RNAII configuration that impairs further elongation of this transcript, thereby reducing the frequency of RNA-DNA duplex formation and initiation of replication. The RNAI-RNAII interaction is counterbalanced by the shorter half-life of RNAI compared to RNAII. The ColE1-encoded Rom protein (also known as Rop) increases the frequency of RNAI-RNAII interactions. The gene for Rom is deleted in many ColE1-based plasmid vectors, resulting in increased copy numbers compared to ColE1 itself. Perturbations of ColE1 plasmid copy number are rapidly mirrored by changes in RNAI concentration, resulting in the enhancement or suppression of replication and the maintenance of ColE1 copy number within a narrow window. 4.3. Rolling-Circle Replication Many small (<10 kbp) plasmids of Gram-positive Eubacteria replicate by a rollingcircle mechanism, which is distinct from the replication of iteron-containing or ColE1like plasmids (see Fig. 3) (47). Rolling-circle plasmids have also been identified in Gram-negative Eubacteria and in Archaea. Some bacteriophage, including M13 of E. coli, also replicate in this way. In rolling-circle replication, binding of a plasmid-encoded replication protein to the leading-strand origin (also known as the double-strand origin) distorts the DNA in this region and exposes a single-stranded region in an extruded cruciform. A nick is introduced at this site by the replication protein and this exposes a 3'OH group from which the leading strand is synthesized by DNA polymerase III. Leading strand initiation differs between rolling circle plasmids, procaryotic chromosomes, and other plasmids, although chain elongation is similar in all systems. As the leading strand is synthesized, the nontemplate strand of the old plasmid is displaced ahead of the replication fork until, eventually, it is removed entirely. The resulting single-stranded intermediate is characteristic of rolling-circle replication and its identification provides evidence that a plasmid replicates by this mechanism (48). The lagging-strand origin (also known as the single-strand origin) is exposed on the displaced single-stranded intermediate and lagging-strand initiation commences at this origin using host replication factors. RNA polymerase synthesizes RNA primers at the lagging strand origin. DNA polymerase I initiates lagging strand synthesis from these RNA primers, after which DNA polymerase III continues elongation. 5. Plasmid Segregation DNA replication produces precise plasmid copies, but plasmids must also ensure that they are distributed to both daughter cells during bacterial cell division. If the Hayes
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.
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.
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.