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.     

Nucleotide sequence of the immunity and lysis region of the ColE9-J plasmid

We have determined the nucleotide sequence of a 1500 bp fragment of the ColE9-J plasmid which encodes colicin E9 immunity and colicin E5 immunity and contains two lys genes. Open reading frames corresponding to the four genes have been located and their position confirmed by transposon mutagenesis of sub-clones of the ColE9-J plasmid. The E9imm gene shows 69% homology at both the nucleotide and the amino acid level to the previously sequenced E2imm gene. The E5imm gene shows little homology to any other E colicin immunity gene which has been sequenced. The lys gene distal to the 3' end of the E5imm gene shows considerable sequence homology to all other previously sequenced E colicin lys genes. The lys gene distal to the 3' end of the E9imm gene is identical to the pColE2 and pColE3 lys genes for the first 59 nucleotides but encodes a much smaller gene product than any other lys gene which has been sequenced. The two lys genes sequenced here are exceptions to Shepherd's rule concerning the number of RNY codons in the three possible reading frames.     

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.     

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.     

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 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.     

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)     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Comparative nucleotide sequences encoding the immunity proteins and the carboxyl-terminal peptides of colicins E2 and E3

Using the M13 dideoxy sequencing technique, we have established the DNA sequences of colicins E2 and E3 which encompass the receptor-binding and the catalytic domains of each of the nucleases, and their immunity (imm) genes. The imm gene of plasmid ColE2-P9 is 255 bp long and is separated from the end of the col gene by a dinucleotide. This gene pair is arranged similarly in plasmid ColE3-CA38 except that the intergenic space is 9 bp and the E3 imm gene is one codon shorter than its E2 counterpart. Comparisons of the E2 and E3 imm sequences indicate considerable divergence whereas the receptor-binding domains of both colicins are highly conserved. The two nuclease domains appear to share some sequence homology. A possible evolutionary relationship between colicin E3 and other microbial extracellular ribonucleases is also suggested from the sequence alignment analysis.     

Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein.

The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322. These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long. An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500. Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein. Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane. Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane. This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.     

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.     

Identification of a unique specificity determinant of the colicin E3 immunity protein.

Plasmid immunity to a nuclease-type colicin is defined by the specific binding of an immunity (or inhibitor) protein, Imm, to the C-terminal nuclease domain, T2A, of the colicin molecule. Whereas most regions of colicin operons exhibit extensive sequence identity, the small plasmid region encoding T2A and Imm is exceptionally varied. Since immunity is essential for the survival of the potentially lethal colicin plasmid (Col), we inferred that T2A and Imm must have co-evolved, retaining their mutual binding specificities. To evaluate this co-evolution model for the col and imm genes of ColE3 and ColE6, we attempted to obtain a stabilized clone from a plasmid which had been destabilized with a non-cognate immunity gene. A hybrid Col, in which the immE3 gene of the ColE3 was replaced with immE6 from ColE6, was lethal to the host cells upon SOS induction. From among this suicidal cell population, we isolated a stabilized, i.e., evolved, clone which produced colicin E3 (E3) stably and exhibited immunity to E3. This change arose from only a single mutation in ImmE6, from Trp48 to Cys, the same residue as in the ImmE3 sequence. In addition, we constructed a series of chimeric genes through homologous recombination between immE3 and immE6. Characterization of these chimeric immunity genes confirmed the above finding that colicins E3 and E6 are mostly distinguished by only Cys48 of the ImmE3 protein.     

Nucleotide sequences from the colicin E8 operon: homology with plasmid ColE2-P9

The primary structures of the immunity (Imm) and lysis (Lys) proteins, and the C-terminal 205 amino acid residues of colicin E8 were deduced from nucleotide sequencing of the 1,265 bp ClaI-PvuI DNA fragment of plasmid ColE8-J. The gene order is col-imm-lys confirming previous genetic data. A comparison of the colicin E8 peptide sequence with the available colicin E2-P9 sequence shows an identical receptor-binding domain but 20 amino acid replacements and a clustering of synonymous codon usage in the nuclease-active region. Sequence homology of the two colicins indicates that they are descended from a common ancestral gene and that colicin E8, like colicin E2, may also function as a DNA endonuclease. The native ColE8 imm (resident copy) is 258 bp long and is predicted to encode an acidic protein of 9,604 mol. wt. The six amino acid replacements between the resident imm and the previously reported non-resident copy of the ColE8 imm ([E8 imm]) found in the ribonuclease-producing ColE3-CA38 plasmid offer an explanation for the incomplete protection conferred by [E8 Imm] to exogenously added colicin E8. Except for one nucleotide and amino acid change in the putative signal peptide sequence, the ColE8 lys structure is identical to that present in ColE2-P9 and ColE3-CA38.     

Genetic analysis of the functional relationship between colicin E3 and its immunity protein.

Partial deletions in the immunity gene of the colicin E3 operon were used to study possible functions of the immunity protein besides protection against exogenous colicin. Nuclease BAL-31 was used to create a series of carboxyl-terminal deletions of the immunity gene. Mutants displaying lowered immunity against exogenous colicin were found, and six that had reduced but detectable levels of immunity were chosen for further analysis. DNA sequence analysis of the deletions showed that all six terminated within the last five codons of the immunity gene. The wild-type immunity gene was replaced by each of the six mutated immunity genes in a plasmid containing an otherwise functional colicin E3 operon. Transformants containing the resulting plasmids produced smaller colonies on solid medium and grew more slowly in liquid culture than transformants carrying the wild-type colicin and immunity genes. This result suggested that immunity protein was required to protect the cell against endogenous colicin E3. This idea was confirmed in experiments in which the colicin E3 and immunity genes were independently cloned on two compatible plasmid vectors.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

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).     

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.     

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.