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)     

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.     

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.     

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.     

Restriction enzyme analysis of the Plasmid ColIb DNA

Summary Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.     

Colicin Ib does not cause plasmid-promoted abortive phage infection of Escherichia coli K-12

Summary A 2.2 kilobase (kb) fragment of ColIdrd-1 cloned in pBR325 causes phage T5 and BF23 infections of Escherichia coli K-12 to abort. This abortive infection is associated with leakage of -galactosidase from the cell. A recombinant plasmid containing a 2.8 kb fragment of ColIdrd-1 encodes colicin Ib but fails to cause abortive infection. Tn5 and Tn10 insertions into ColIdrd-1 that abolish colicin Ib production have no effect on the abortive infection phenotype. These findings are inconsistent with a previously proposed role for colicin Ib in causing phage infections to abort.     

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.     

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.     

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     

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

Summary Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).     

Nucleotide sequence of a DNA fragment that contains the abi gene of the ColIb plasmid

A 1989-bp PstI DNA fragment from the ColIb plasmid, which contains the abi gene that is necessary for the abortive response to infections by bacteriophage BF23 or T5, was sequenced. A candidate open reading frame for the abi gene has been suggested on the basis of a Shine-Dalgarno sequence appropriately placed ahead of its ATG initiation codon, a promoter upstream from the Shine-Dalgarno sequence, and a location compatible with deletion mapping. The polypeptide that would be coded by this open reading frame is 89 amino acids long and strongly hydrophobic. A promoter that could serve this open reading frame was detected by exonuclease III "footprinting" using RNA polymerase from uninfected Escherichia coli as the DNA-binding protein.     

Cell wall lipopolysaccharide response to the ColIb plasmid mutants.

Mutants of ColIb plasmid affected the synthesis of O-side chains of lipopolysaccharides (LPS) in Salmonella. The plasmid srd 25 (defective in colicin synthesis) caused a significant decline of rhamnose and mannose content and lack of abequose in LPS of S. typhimurium. The number of repeating units in O-side chains was decreased after the indroduction of srd 25. Cultures of S. typhimurium and S. enteritidis harboring drd2 (derepressed in colicin production) polymerised dideoxyhexose-defective O-side chains i.e. deprived of abequose and tyvelose, respectively. In dideoxyhexoseless S. meleagridis the content of rhamnose and mannose were reduced. The information for the alterations of Salmonella LPS was contained in the plasmid genome. In the wild-type plasmids the genes controlling the O-antigen changes were not expressed.     

Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

DNA primase of plasmid ColIb is involved in conjugal DnA synthesis in donor and recipient bacteria.

The sog gene of the IncI alpha group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sog- plasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog- plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material.     

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.     

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.     

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.     

Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9

The IncI₁ plasmid ColIb-P9 is among a group of related plasmids that encode the I₁ type of conjugation system. The I₁ system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I₁ conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.     

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

Summary R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod^-) or nitrogen fixation (Fix^-) have been readily obtained. In some Nod^- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. ³²P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod^- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod^- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod^- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.