Characterization of a mini-ColC1 plasmid.

An in vitro constructed plasmid, pVH15, consisting of the entire genome of the plasmid ColE1, the tryptophan operon of Escherichia coli, and regions of the bacteriophage PHI80pt190, spontaneously gave rise in E. coli to a mini-ColE1 plasmid consisting of approximately one-half of the ColE1 genome and a small segment of phi80pt190 DNA. This mini-ColE1 plasmid, designated pVH51, has a molecular weight of approximately 2.1 X 10(6) and possesses a single EcoRI restriction site. Heteroduplex analyses showed that about 90% of the pVH51 plasmid hybridizes to about 50% of the ColE1 plasmid. Phenotypically, pVH51 did not produce colicin E1 but conferred immunity to this colicin. The number of mini-ColE1 plasmid molecules per cell was maintained at a four- to fivefold higher level than normal ColE1. A mini-ColE1 hybrid plasmid, designated pML21 and consisting of pVH51 and the kan fragment of plasmid pSC105 inserted at the EcoRI restriction site of mini-ColE1, was maintained at a lower copy number level than pVH51. As in the case of normal ColE1, both pVH51 and pML21 continued to replicate in the presence of chloramphenicol. The promotion of conjugal transfer of pVH51 and pML21 by a self-transmissible plasmid was greatly reduced compared with normal ColE1.     

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

Dependence of the activity of phi X 174 B-promoter in the expression of Escherichia coli gal-operon on the number of its copies and their orientation

Promoter activities of different restriction fragments of the R8 DNA region of phage phi X 174 were compared. The studied DNA fragments included HindII fragment R8 (B-promoter), its left portion 49 nucleotide long, and the central segment containing 113 nucleotides generated by AluI. The promoter activity of these fragments was quantitated by the appearance of uridyltransferase and galactokinase activities in Escherichia coli clones carrying plasmids pHD68-17. The gal-promoters of these plasmids was substituted for the three aforementioned restriction fragments. The R8 region and its central part (BII-promoter) had comparable promoter activities while the left part containing the putative BI-promoter, did not induce clones with the expressed gal-operon. Clones containing 1, 2, 3 copies of the promoter fragment R8 were selected. No clones were revealed with more copies. All selected di- and tri-promoter clusters in plasmids had the same correct orientation of all inserted promoters with respect to the gal-operon. The expression of the gal-operon in E. coli was nearly directly proportional to the number of the phi X 174 B-promoters inserted before the operon.     

The transposon Tn1 as a probe for studying ColE1 structure and function.

Summary Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be -lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.     

Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13.

The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined. Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene. The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities. On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons. The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein. The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13. Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13).     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

Incompatibility between E colicin plasmids

We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line. Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J. Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.     

An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Excision of a DNA sequence determining kanamycin resistance from a ColE1-Km recombinant plasmid

Summary A 4.8×10⁶ dalton ECoRI-generated fragment of the R-factor R6-5 carrying the gene for kanamycin resistance (Km) was joined in vitro to ECoRI-treated ColE1 plasmid DNA. Transformation ofE. coli with the ColE1-Km recombinant plasmid yielded clones, which were immune to colicin E1, resistant to kanamycin and failed to produce colicin E1. During multiplication of this recombinant plasmid in the presence of chloramphenicol, cells expressed an increased resistance to kanamycin. Transformation studies with the recombinant DNA molecule showed very frequent loss of Km resistance in those cells harbouring a preexisting F'gal plasmid. Since colicin immunity is not affected and the col^- phenotype is still present, one has to test for a remaining DNA sequence further existing in ColE1 DNA by cleaving the plasmid DNA with the ECoRI restriction endonuclease. The full length of ColE1 DNA (6.2 kb) was restored, which confirmed that no deletion of ColE1 DNA sequences had occured. The remaining DNA sequence was identified as a 2.0 or 2.2 kb segment. On the basis of the length of the excised fragment it is proposed that the insertion sequence IS1 and a part of the inverted repeat sequence with coordinates 21.0 to 22.0 of the R6-5 DNA are recognised by a nucleolytic function.     

A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages.

A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, low-molecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.     

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.     

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.