Cloning and nucleotide sequence of the ColIb shufflon

The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.     

DNA-independent transport of plasmid primase protein between bacteria by the I1 conjugation system

The ColIb-P9 (IncI1)-encoded conjugation system supports transfer of the plasmid T-strand plus hundreds of molecules of the Sog polypeptides determined by the plasmid primase gene. Here, we report that Sog primase is abundantly donated to the recipient cell from cells carrying a non-transferable ColIb plasmid deleted of the nic site essential for DNA export. Such DNA-independent secretion of Sog primase is typical of authentic conjugation, both in being blocked when the recipient cell specifies the entry exclusion function of ColIb and in requiring the thin I1 pilus encoded by the ColIb pil system under the mating conditions used. It is proposed that Sog polypeptides form a complex with the ColIb T-strand during conjugation and aid DNA transport through processive secretion of the proteins into the recipient cell. Functional and genetic relationships between the ColIb conjugation system and other type IV secretion pathways are discussed.     

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.     

Inflammation Fuels Colicin Ib-Dependent Competition of Salmonella Serovar Typhimurium and E. coli in Enterobacterial Blooms

The host''s immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae (“inflammation-inflicted blooms”). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.     

The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.     

The IncI plasmids R144, R64 and ColIb belong to one exclusion group

The exclusion relationship between the IncI plasmids R144, R64 and ColIb was studied in such a way that incompatibility interference was avoided. Genetic crosses with an R144-derived Hfr donor, crosses with recipient strains carrying R144-derived exclusion genes on a recombinant plasmid compatible with R144, and measurement of transmission frequencies of a recombinant plasmid compatible with IncI plasmids after mobilization by R144 revealed that R144, R64 and ColIb belong to one exclusion group.     

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.     

Regulation of Bacteriophage T5 Development by ColI Factors

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb(+) cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb(+) cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis.     

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.     

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.     

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.     

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.     

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)     

IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions.

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.     

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 sequence of the gene (ard) encoding the antirestriction protein of plasmid colIb-P9.

The IncI1 plasmid ColIb-P9 was found to encode an antirestriction function. The relevant gene, ard (alleviation of restriction of DNA), maps about 5 kb from the origin of transfer, in the region transferred early during bacterial conjugation. Ard inhibits both restriction and modification by each of the four type I systems of Escherichia coli tested, but it had no effect on restriction by either EcoRI, a type II system, or EcoP1, a type III system. The nucleotide sequence of the ColIb ard gene was determined; the predicted molecular weight of the Ard polypeptide is 19,193. The proposed polypeptide chain contains an excess of 25 negatively charged amino acids, suggesting that its overall character is very acidic. Deletion analysis of the gene revealed that the Ard protein contained a distinct functional domain located in the COOH-terminal half of the polypeptide. We suggest that the biological role of the ColIb Ard protein is associated with overcoming host-controlled restriction during bacterial conjugation.     

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.     

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides

Summary Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effects were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.