Bidirectional replication of the mini-ColE1 plasmid pVH51.

Replicating molecules of the min-ColE1 plasmid pVH51 have been examined by electron microscopy after cleavage with the restriction endonuclease EcoRI. Replication apparently starts at a unique site indistinguishable from the origin of replication used by the parental plasmid ColE1. In contrast to ColE1, the structure of the majority of the replication intermediates was consistent with a bidirectional mode of replication. A minor portion of the molecules appeared to replicate unidirectionally in either direction from the same origin.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.

The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.     

New mini-ColE1 as a molecular cloning vehicle.

A new mini-ColE1 plasmid, designated pAC105, was isolated. It has a molecular weight of 1.6 X 10(6) and carries information for its self-replication as well as information for conferring colicin E1 immunity upon its host. Furthermore, pAC105 undergoes replication in the presence of chloramphenicol even when a foreign deoxyribonucleic acid (pSC101) is inserted into its single EcoRI restriction site. Studies in minicell-producing strains demonstrate that pAC105 codes for only two or three polypeptides of low molecular weight. The advantages of using it as a molecular cloning vehicle are discussed.     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

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.     

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.     

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.     

Characterization of the ColE9-J plasmid and analysis of its genetic organization

We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.     

Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids

We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector. By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity. In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment. Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity. Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion. We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter. The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.     

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.     

Trimethoprim-induced DNA polymerase I deficiency in Escherichia coli K-12

Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase. Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain. These colonies therefore had PolA- phenotypes. Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them. Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.     

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.     

Trimethoprim-Produced F-Specific Insertion Mutations in Escherichia coli K-12

Besides producing thymine-requiring mutants (thy), trimethoprim (TMP) cured the mini-ColE1 replicon pML21 at an appreciable frequency. The cured Escherichia coli K-12 cells behaved like polA mutants by failing to support the stable maintenance of the ColE1 plasmid. The mini-F replicon pSC138, which was lacking all three insertion sequences (IS3, gammadelta, and IS2) normally used for F-specific integration and excision, was not cured by TMP. Instead, it integrated into specific regions of the E. coli chromosome and thus caused auxotrophic mutations in operons which were always localized on either side of oriC (origin of chromosomal replication). The incompatibility and replication functions of the integrated plasmid in auxotrophs were retained, and the plasmid DNAs recovered from spontaneously occurring revertants did not show any alterations in their contour lengths as determined by electron microscopy. The F replicon (fragment 5) contained in plasmid pSC138 carried two origins of replication, the primary origin, oriV(1) at 42.6F and the secondary origin, oriV(2), at 44.1F. Another mini-F plasmid pMF21, deleted of the primary origin of replication (oriV(1)), was still capable of autonomous replication but failed to integrate onto the chromosome after TMP treatment. Furthermore, the composite plasmid pRS5, which normally uses only the replication origin and functions of the pSC101 component, was also insensitive to TMP. On the basis of these results, we propose a new scheme of F integration via the functional oriV(1) and suggest the involvement of a similar mechanism in the formation of Hfr strains by integrative suppression.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.