Construction and analysis of miniplasmids of the colicin Ib plasmid

Miniplasmids of the colicin Ib (ColIb) plasmid have been isolated from two Tn5-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.     

alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

We performed three types of experiments to test the hypothesis that abortive infection of T5 bacteriophage in Escherichia coli (ColIb+) is due to internally released colicin. (i) We measured the sensitivity of cells to colicin under a variety of conditions and then looked at the plating efficiency of T5 in ColIb+ cells under these same conditions. Cells grown at 42 degrees C or with hexanol had a reduced sensitivity to externally added colicin and an increased efficiency for T5 when the ColIb plasmid was present in the infected cells. Phage growth was far from normal, however. (ii) We measured the colicin sensitivity of a mutant bacterium that grew T5 normally even in the presence of the ColIb plasmid and measured the plating efficiency of T5 on another mutant that was colicin tolerant. Here again, the correlation between colicin activity and inhibition of phage replication was not complete. (iii) We looked for colicin-negative plasmid mutants and tested the ability of cells containing these plasmids to support the growth of T5. These experiments used Tn5, a kanamycin resistance transposon, as the mutagen. All possible combinations of colicin production and phage inhibition were found, including mutants that produced no colicin but still inhibited phage production.     

The relationship of the delta transfer factor and KColIb to the ColIb plasmid

The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared. Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids. The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid. It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments. However, ColIb, KColIb and delta were homologous for at least 70% of their lengths. The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb. In addition, delta and KColIb differed from ColIb at similar sites. The possible evolution of these plasmids is discussed.     

ColIb plasmid genes that inhibit the replication of T5 and T7 bacteriophage

The colicin Ib (ColIb) plasmid genes that inhibit the replication of the T5-like and T7 bacteriophage have been cloned on an approximately 7200-bp ClaI fragment and their sites relative to each other and to the colicin immunity (imm) gene have been mapped. The inhibition of wild-type T7 by the clone is shown to be caused by the same gene or genes (pic) that cause the inhibition of T7 kinase-negative mutants and is a different gene than the one that causes inhibition of T5 (ibf or abi). The pic gene does not hybridize to the pif genes of the F plasmid that also cause the replication of T7 to be inhibited. The abi gene and the pic gene map very closely together but are under the control of different promoters. The abi gene has a maximum size of 900 bp and lies approximately 3000 bp away from the immunity gene, distal to the colicin gene. A site which maps in or near the gene binds very tightly to Escherichia coli RNA polymerase. The pic gene or genes lie between the abi gene and the imm gene and are contiguous with abi. Promoters for pic have been mapped and hypotheses to explain the inhibition of T7 by a cloned gene but not the whole ColIb plasmid are presented.     

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.     

Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.     

Is abortive infection by bacteriophage BF23 of Escherichia coli harboring ColIb plasmids due to cell killing by internally liberated colicin Ib?

Infection of Escherichia coli harboring ColIb+ plasmids with bacteriophage BF23+ is abortive and resulted in changes of membrane permeability as measured by efflux of nucleotides and K+. A single pre-early gene product of BF23+ was necessary and sufficient to elicit the abortive response. Appropriate mutations in this pre-early gene allowed a productive infection in ColIb+ cells. Appropriate mutations in the ColIb plasmid also allowed a productive infection with BF23+. A comparison of changes occurring during abortive infection and during killing of sensitive cells by external colicin Ib or Ia, together with certain genetic data, has led to the conclusion that membrane changes accompanying the two phenomena are the result of a common mechanism, namely, the interaction of free colicin with the cytoplasmic membrane.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

Inhibition of growth of bacteriophage BF23 by the ColIb plasmid: Identification of the ibfA and ibfB genes of the ColIb plasmid

Summary A 3.7 Mdal DNA fragment of plasmid ColIb which carried all the genetic information determining the growth inhibition of bacteriophage BF23 (Ibf phenotype) but not for production of colicin Ib protein (Cib) and immunity to colicin Ib (Imm) was cloned in the pBR322 vector. We also cloned the 8.7 Mdal DNA fragment that was responsible for both Cib and Imm but not for Ibf phenotype. Thus, these results clearly showed that the gene(s) determining Ibf are different from those for Cib or Imm. Dissection of the Ibf-DNA revealed that at least two genes, ibfA and ibfB, were involved in the Ibf phenotype. The ibfA gene was mapped within a 1.45 Mdal EcoRI DNA fragment (E-13); mutation of this gene by deletion or by insertion of Tn5, a kanamycin resistance transposon, resulted in the complete loss of Ibf phenotype. The ibfB gene, mapped around a 0.32 Mdal HindIII DNA fragment (H-7), was found to be active in trans to ibfA, and its function seemed to promote ibfA activity. The genetic map of the ibf genes in relation to other ColIb markers was determined as ibfB-ibfA-imm-cib. Attempts to identify the ibfA gene product in the minicell system, however, did not succeed.Preliminary results were presented in the 1980 Annual Meeting of the Japan Molecular Biology Society. (Uemura and Mizobuchi Abst Ann Mol Meet 1980, p 35)     

Cloning of immunity and structural genes for colicin V

The colicin V immunity and structural genes of plasmid pColV-B188 were cloned into the vectors pMB9, pBR322, and pMK16. Both genes are closely linked and can be isolated on a 900-base-pair deoxyribonucleic acid fragment. Insertion of the transposon Tn5 into this cloned sequence led to the construction of a mutant plasmid which conferred colicin V immunity, but not the ability to produce this colicin. Analysis of the products determined by these cloned genes in cells has led to the conclusion that the polypeptide involved in immunity has a molecular weight of about 6,500, whereas the colicin has a molecular weight of approximately 4,000.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

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.     

Four plasmid genes are required for colicin V synthesis, export, and immunity.

The colicin V production and immunity genes were isolated from plasmid pColV-K30. A HindIII-to-SalI fragment of 9.4 kilobases was cloned into the compatible vectors pBR322 and pACYC184. Mutants defective in colicin production were generated by Tn5 insertions and by constructing deletions in vitro. Physical analysis of these mutations identified a 4.4-kilobase region of this DNA which contains all the plasmid genes (cva) needed for the production of colicin V. The colicin V immunity determinant (cvi) is in a 700-base-pair fragment located within one end of this region. Complementation tests identified three genes, called cvaA, cvaB, and cvaC, required for colicin production. Analysis of the proteins labeled in minicells harboring various Tn5 insertions allowed us to identify protein products for the cvaA and cvaC genes. Mutations in cvaA and cvaB eliminated colicin activity in culture supernatants, but not within the cells. Mutations in cvaC, however, eliminated all detectable activity. From these results we conclude that the cvaC gene codes for the structural gene for colicin V, while cvaA and cvaB are apparently needed for the normal export of the colicin.     

Isolation and genetic characterizaion of Escherichia coli K-12 mutations affecting bacteriophage T5 restriction by the ColIb plasmid.

A mutant derivative of Escherichia coli K-12 has been isolated which is permissive for bacteriophage T5 infection even when harboring a wild-type ColIb plasmid. The fully permissive phenotype was the result of two mutations that are located near the rpsL-rpsE region on the E. coli chromosome and are recessive to the wild-type alleles. These mutations had little or no effect on induction of colicin synthesis and did not affect the expression of antibiotic resistance by the resistance plasmids R64drd11 or R1drd19. Cells harboring the mutant alleles grew more slowly than isogenic wild-type derivatives in either minimal or complete media.     

Tn1 insertion in Col Ib plasmid

Eight clones ColIb::Tn1 were isolated by means of transformation of E. coli 921 with DNA ColIb, RP4. Linkage between markers controlling ampicillin-resistance and synthesis of colicine was found in conjugation and transduction experiments.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

Analysis of a cloned colicin Ib gene: complete nucleotide sequence and implications for regulation of expression.

The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed.     

A hisT::Tn5 mutation affects production of microcins B17, C7, and H47 and colicin V.

A Tn5 insertion decreasing the production of microcin B17 was mapped to 50.2 min on the Escherichia coli chromosome map. Sequence analysis showed that the insertion disrupted hisT, the gene encoding pseudouridine synthase I, a tRNA-modifying enzyme. hisT::Tn5 mutant cells were also shown to be defective for the production of other antibiotic peptides, such as microcin C7, microcin H47, and colicin V.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)