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Bai Y, Zhang YL, Wang JD, Zhang ZS, Zhou DY. Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity. World J Gastroenterol 2004; 10:2498-502. [PMID: 15300892 PMCID: PMC4572149 DOI: 10.3748/wjg.v10.i17.2498] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity.
METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.
RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 × 1010 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.
CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.
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Affiliation(s)
- Yang Bai
- PLA Institute for Digestive Medicine, Nanfang Hospital, the First Military Medical University, Guangzhou 510515, Guangdong Province, China.
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2
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Oscarsson J, Westermark M, Löfdahl S, Olsen B, Palmgren H, Mizunoe Y, Wai SN, Uhlin BE. Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars typhi and paratyphi A. Infect Immun 2002; 70:5759-69. [PMID: 12228306 PMCID: PMC128311 DOI: 10.1128/iai.70.10.5759-5769.2002] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli. Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyA(STy) and ClyA(SPa), show >/=90% amino acid identity to E. coli cytolysin A, ClyA(EC), and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyA(EC). The chromosomal clyA(STy) gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyA(STy) reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyA(STy) chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyA(STy) gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified.
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Affiliation(s)
- Jan Oscarsson
- Department of Molecular Biology, Umeå University, S-90187 Umeå Swedish Institute for Infectious Disease Control, S-17182 Solna, Sweden
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3
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Rasmussen TA, Nolan JM. G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme. Gene 2002; 294:177-85. [PMID: 12234679 DOI: 10.1016/s0378-1119(02)00766-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.
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Affiliation(s)
- Terri A Rasmussen
- Department of Biochemistry-SL43, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699, USA
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4
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Ingmer H, Miller C, Cohen SN. The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response. Mol Microbiol 2001; 42:519-26. [PMID: 11703672 DOI: 10.1046/j.1365-2958.2001.02661.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.
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Affiliation(s)
- H Ingmer
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA
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5
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Miller C, Cohen SN. Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA. J Bacteriol 1999; 181:7552-7. [PMID: 10601213 PMCID: PMC94213 DOI: 10.1128/jb.181.24.7552-7557.1999] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.
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Affiliation(s)
- C Miller
- Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA
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6
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Abstract
A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed. These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures. The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by alpha complementation. These vectors have proven useful for a variety of applications where conditional replication of a recombinant plasmid is desired.
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Affiliation(s)
- G J Phillips
- Department of Microbiology, Iowa State University, Ames, Iowa, 50011,
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7
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Ingmer H, Cohen SN. Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning. J Bacteriol 1993; 175:7834-41. [PMID: 8253672 PMCID: PMC206959 DOI: 10.1128/jb.175.24.7834-7841.1993] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division.
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Affiliation(s)
- H Ingmer
- Department of Genetics, Stanford University School of Medicine, California 94305-5120
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8
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Abstract
The incompatibility that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superhelicity, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid. We propose a model by which the par locus can enhance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.
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Affiliation(s)
- C A Miller
- Department of Genetics, Stanford University School of Medicine, California 94305
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9
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Olsén A, Arnqvist A, Hammar M, Sukupolvi S, Normark S. The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli. Mol Microbiol 1993; 7:523-36. [PMID: 8459772 DOI: 10.1111/j.1365-2958.1993.tb01143.x] [Citation(s) in RCA: 226] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Curli encoded by the curlin subunit gene, csgA, are fibronectin- and laminin-binding fibres expressed by many natural Escherichia coli and E. coli K-12 strains in response to low temperature, low osmolarity and stationary-phase growth conditions. Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase-inducible genes. Many commonly used K-12 strains carry an amber mutation in rpoS. Strains able to form curli carry an amber suppressor whereas curli-negative E. coli K-12 strains, in general, are sup0. Introduction of SupD, SupE, or supF suppressors into sup0 strains resulted in expression of temperature-regulated curli. In curli-deficient, RpoS- E. coli K-12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone-like protein H-NS. Curli expression, fibronectin binding, and csgA transcription remain temperature- and osmoregulated in such double mutants. Our data suggest that RpoS+ strains, and hence curli-proficient strains of E. coli K-12, are relieved for the transcriptional repression mediated by the H-NS protein upon accumulating RpoS as cells reach stationary phase.
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Affiliation(s)
- A Olsén
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
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10
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Pyne C, Bognar AL. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro. J Bacteriol 1992; 174:1750-9. [PMID: 1548226 PMCID: PMC205775 DOI: 10.1128/jb.174.6.1750-1759.1992] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.
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Affiliation(s)
- C Pyne
- Department of Microbiology, University of Toronto, Ontario, Canada
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11
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Tadayyon M, Broome-Smith JK. TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins. Gene X 1992; 111:21-6. [PMID: 1312501 DOI: 10.1016/0378-1119(92)90598-j] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.
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Affiliation(s)
- M Tadayyon
- Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, U.K
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12
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Rasmussen LJ, Møller PL, Atlung T. Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli. J Bacteriol 1991; 173:6390-7. [PMID: 1917868 PMCID: PMC208971 DOI: 10.1128/jb.173.20.6390-6397.1991] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.
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Affiliation(s)
- L J Rasmussen
- Department of Microbiology, Technical University of Denmark, Copenhagen
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13
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Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus. J Biol Chem 1991. [DOI: 10.1016/s0021-9258(18)54871-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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14
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Kimlova LJ, Pyne C, Keshavjee K, Huy J, Beebakhee G, Bognar AL. Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli. Arch Biochem Biophys 1991; 284:9-16. [PMID: 1989505 DOI: 10.1016/0003-9861(91)90254-g] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.
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Affiliation(s)
- L J Kimlova
- Department of Microbiology, University of Toronto, Ontario, Canada
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15
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Waugh DS, Pace NR. Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria. J Bacteriol 1990; 172:6316-22. [PMID: 1699929 PMCID: PMC526815 DOI: 10.1128/jb.172.11.6316-6322.1990] [Citation(s) in RCA: 57] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
We report the construction of a strain of Escherichia coli in which the only functional gene for the RNA moiety of RNase P (rnpB) resides on a plasmid that is temperature sensitive for replication. The chromosomal RNase P RNA gene was replaced with a chloramphenicol acetyltransferase gene. The conditionally lethal phenotype of this strain was suppressed by plasmids that carry RNase P RNA genes from some distantly related eubacteria, including Alcaligenes eutrophus, Bacillus subtilis, and Chromatium vinosum. Thus, the rnpB genes from these organisms are capable of functioning as the sole source of RNase P RNA in E. coli. The rnpB genes of some other organisms (Agrobacterium tumefaciens, Pseudomonas fluorescens, Bacillus brevis, Bacillus megaterium, and Bacillus stearothermophilus) could not replace the E. coli gene. The significance of these findings as they relate to RNase P RNA structure and function and the utility of the described strain for genetic studies are discussed.
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Affiliation(s)
- D S Waugh
- Department of Biology, Indiana University, Bloomington 47405
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16
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Abstract
Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations. We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101. Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons. topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems. Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmid's inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects.
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Affiliation(s)
- C A Miller
- Department of Genetics, Stanford University School of Medicine, California 94305-5120
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17
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Hwang YW, Sanchez A, Miller DL. Mutagenesis of bacterial elongation factor Tu at lysine 136. J Biol Chem 1989. [DOI: 10.1016/s0021-9258(18)83183-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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18
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Wei D, Parulekar SJ, Stark BC, Weigand WA. Plasmid stability and α-amylase production in batch and continuous cultures ofBacillus subtilis TN106[pAT5]. Biotechnol Bioeng 1989; 33:1010-20. [DOI: 10.1002/bit.260330810] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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19
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Lund B, Marklund BI, Strömberg N, Lindberg F, Karlsson KA, Normark S. Uropathogenic Escherichia coli can express serologically identical pili of different receptor binding specificities. Mol Microbiol 1988; 2:255-63. [PMID: 2898091 DOI: 10.1111/j.1365-2958.1988.tb00027.x] [Citation(s) in RCA: 139] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Uropathogenic Escherichia coli frequently express P-pilus adhesins that recognize Gal alpha (1-4)Gal-containing glycoconjugates. The P-pilus adhesin of the E. coli isolate J96 is encoded by the pap gene cluster and has been shown to agglutinate P1-erythrocytes. We now describe a novel gene cluster from J96, prs, which is responsible for the agglutination of sheep erythrocytes. The structurally related gene clusters both expressed pili exhibiting the F13 antigen. Analysis of mutants of cloned prs sequences, together with trans-complementation of pap and prs genes, identified the sheep-specific adhesin as the 37-kD PrsG protein. The prsG gene occupies the equivalent position in prs as occupied by papG, which specifies the Gal alpha (1-4)Gal-specific adhesin of pap. PrsG was shown to be structurally distinct from PapG since PapG-specific antiserum did not cross-react with PrsG. Using a solid phase glycolipid receptor binding assay, PrsG was found to specify preferential binding to the Forssman antigen, a major constituent of sheep erythrocyte membranes. The binding epitope was identified as the GaINAc alpha (1-3)GaINAc moiety. This is the first direct evidence that serologically identical pili may present antigenically distinct adhesins, each capable of binding to a specific receptor.
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Affiliation(s)
- B Lund
- Department of Microbiology, University of Umeå, Sweden
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20
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Heacock PN, Dowhan W. Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli. J Biol Chem 1987. [DOI: 10.1016/s0021-9258(18)45164-2] [Citation(s) in RCA: 30] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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21
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Nilsson G, Belasco JG, Cohen SN, von Gabain A. Effect of premature termination of translation on mRNA stability depends on the site of ribosome release. Proc Natl Acad Sci U S A 1987; 84:4890-4. [PMID: 2440033 PMCID: PMC305211 DOI: 10.1073/pnas.84.14.4890] [Citation(s) in RCA: 98] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed. Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments. Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA. These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation. However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript.
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22
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Abstract
It has been postulated that deletions mediated by transposable elements are intramolecular transposition events. An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication. We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons. We show that when two origins (ori's) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions. On the contrary, when two ori's (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions. However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions.
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23
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Michiels T, Cornelis G. Tn951 derivatives designed for high-frequency plasmid-specific transposition and deletion mutagenesis. Gene 1986; 43:175-81. [PMID: 3017811 DOI: 10.1016/0378-1119(86)90205-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
We describe the construction of a system allowing high-frequency transposition and deletion mutagenesis with class-II transposons containing a kanamycin or a chloramphenicol-resistance marker. The system utilizes the transposition function of Tn3 and the resolution function of Tn951/Tn2501 which leads to an uncoupling of the resolution and repression functions. It consists of defective transposons inserted into conjugative, replication thermosensitive plasmids. The properties of the system are: easily selectable resistance markers, high transposition frequencies onto plasmids, low transposition frequencies onto the host chromosome, placement of the tnpA gene outside the transposons so that "second-generation" transposition does not occur, possibility to transpose the whole system onto other plasmid vectors with different selection strategies, consecutive use of two transposons for deletion mutagenesis and restriction mapping.
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24
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Winans SC, Walker GC. Identification of pKM101-encoded loci specifying potentially lethal gene products. J Bacteriol 1985; 161:417-24. [PMID: 3881396 PMCID: PMC214887 DOI: 10.1128/jb.161.1.417-424.1985] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype).
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25
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Jasin M, Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol 1984; 159:783-6. [PMID: 6086588 PMCID: PMC215717 DOI: 10.1128/jb.159.2.783-786.1984] [Citation(s) in RCA: 122] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.
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26
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Abstract
Three distinct segments (the partition-related, or PR segments) within the 370 bp par region of pSC101 have been shown by deletion analysis to be involved in partitioning of the plasmid to daughter cells. The two lateral segments are direct repeats, each of which potentially can pair with an inverted repeat located between them to form a hairpin-loop structure. Deletion of either lateral segment, together with the middle segment, results in plasmid instability (the Par- phenotype). Deletion of one PR segment yields a stable plasmid that nevertheless shows reduced ability to compete with a coexisting wild-type derivative of the same replicon (the Cmp- phenotype). Deletion of all three segments results in a rate of plasmid loss far in excess of that predicted from the observed copy number of the plasmid. Analysis of the segregation properties of these mutants and of temperature-sensitive and high copy number derivatives of the pSC101 replicon suggests a model in which the par function allows the nonreplicating plasmids of the intracellular pool to be counted as individual molecules, and to be distributed evenly to daughter cells. In the absence of par, the multicopy pool of plasmids behaves as a single segregation unit.
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27
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Jasin M, Regan L, Schimmel P. Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site. Cell 1984; 36:1089-95. [PMID: 6200234 DOI: 10.1016/0092-8674(84)90059-x] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Recent data suggest that size polymorphism of aminoacyl tRNA synthetase is due to variable fusions of additional functional domains to a catalytic core so that, in a large synthetase, a substantial part of the polypeptide is dispensable for catalytic activity. We demonstrate here that a dispensable domain, joined to the catalytic core of a large synthetase, can activate the catalytic sites. This is shown by complementation of an activity-deficient mutant enzyme by protein fragments that contain internal deletions within the catalytic domain and are themselves devoid of activity. The complementation is dependent upon the presence of a defined segment of polypeptide that is remote in the sequence from the catalytic core. Substantial coupling has been established between dispensable and indispensable component pieces. This could be a mechanism to build efficiently large enzymes which integrate the catalytic sites with other previously shown functional roles.
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28
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Linder P, Churchward G, Caro L. Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000. J Mol Biol 1983; 170:287-303. [PMID: 6313942 DOI: 10.1016/s0022-2836(83)80149-1] [Citation(s) in RCA: 39] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.
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29
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Riise E, Stougaard P, Bindslev B, Nordström K, Molin S. Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1. J Bacteriol 1982; 151:1136-45. [PMID: 7050078 PMCID: PMC220389 DOI: 10.1128/jb.151.3.1136-1145.1982] [Citation(s) in RCA: 35] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.
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30
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Curiale MS, Levy SB. Two complementation groups mediate tetracycline resistance determined by Tn10. J Bacteriol 1982; 151:209-15. [PMID: 6282805 PMCID: PMC220228 DOI: 10.1128/jb.151.1.209-215.1982] [Citation(s) in RCA: 75] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The structural and regulatory tetracycline resistance genes of transposon Tn10 are located on a 2,700-base pair HpaI fragment. We have used eight tetracycline-sensitive mutations in the 2,700-base pair fragment, cloned into two compatible plasmids, to demonstrate that two complementation groups are required for tetracycline resistance. By genetic recombination with plasmids containing the regulatory or structural regions for resistance, we have determined that both complementation groups reside within the structural region. The complementation groups, designated tetA and tetB, are proximal and distal, respectively, to the promoter for the tetracycline resistance structural region. The tetB mutations are in the portion of the structural region that is known to encode the 36,000-molecular-weight, inner-membrane TET protein. The levels of tetracycline resistance expressed during complementation suggest a complex interaction between the products of the tetA and tetB loci.
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31
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Sninsky JJ, Uhlin BE, Gustafsson P, Cohen SN. Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli. Gene 1981; 16:275-86. [PMID: 7044891 DOI: 10.1016/0378-1119(81)90083-4] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.
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32
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Cornelis G, Sommer H, Saedler H. Transposon Tn951 (TnLac) is defective and related to Tn3. MOLECULAR & GENERAL GENETICS : MGG 1981; 184:241-8. [PMID: 6276693 DOI: 10.1007/bf00272911] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene. In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition. Transposition of Tn951 duplicates 5 bp of target DNA sequence.
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33
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Cesareni G. The target of the negative regulator of pMB1 replication overlaps with part of the repressor coding sequence. MOLECULAR & GENERAL GENETICS : MGG 1981; 184:40-5. [PMID: 7038387 DOI: 10.1007/bf00271192] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Plasmid mutants (svir), insensitive to inhibition by the repressor of initiation of pMB1 replication, have been selected by exploiting their ability to support lambda growth in the presence of lambda repressor and inhibitor of plasmid replication. The alteration in the mechanism that controls plasmid replication causes a change in the plasmid copy number, svir mutants are dominant, as expected for mutants in the target of a repressor, but at the same time they are unable to synthesise a repressor active on the wild-type target. This lack of cross interaction between svir mutants and a co-resident wild-type plasmid results in their compatibility. These findings are explained by postulating that the target of the inhibitor of pMB1 replication coincides with part of the DNA segment that codes for the inhibitor itself. As a consequence single base pair changes in the target result in altered repressor molecules.
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34
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Light J, Molin S. Replication control functions of plasmid R1 act as inhibitors of expression of a gene required for replication. MOLECULAR & GENERAL GENETICS : MGG 1981; 184:56-61. [PMID: 7038389 DOI: 10.1007/bf00271195] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
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35
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Stougaard P, Molin S, Nordström K. RNAs involved in copy-number control and incompatibility of plasmid R1. Proc Natl Acad Sci U S A 1981; 78:6008-12. [PMID: 6171808 PMCID: PMC348966 DOI: 10.1073/pnas.78.10.6008] [Citation(s) in RCA: 118] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Replication of plasmid R1 is controlled by the products of two genes, copA and copB, that act as inhibitors of replication. Here it is shown that one small RNA synthesized from the copA gene acts as replication inhibitor. This RNA molecule was identified from analyses of RNAs synthesized in EScherichia coli minicells carrying R1 miniplasmids or chimeric plasmids containing the copA gene. In minicells, This RNA was found to be unstable with a half-life of less than a few minutes. Two mutant hybrid plasmids lacking the inhibitor function did not express the RNA normally made from plasmids carrying the wild-type copA allele. Nucleotide sequence analysis of one of the copA mutants showed that a base substitution had occurred within the promoter sequence in front of the copA gene. DNA sequence analysis of the other mutant showed that a putative transcription-termination sequence was affected. The DNA sequence analysis also showed that the RNA molecule synthesized from the copA gene is untranslatable but has the potential for a high degree of secondary structure.
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36
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Abstract
Recently a new insertion element (IS102)b ha been described in plasmid pSC101. We have determined its complete sequence: it consists of 1057 bp; 338 bp at one end are identical to those already determined for the kanamycin resistance transposon Tn903. It is not flanked by any direct repeat. Its coding capabilities are discussed, and compared to those of IS903.
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37
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Taylor DE, Levine JG, Bradley DE. In vivo formation of a plasmid cointegrate expressing two incompatibility phenotypes. Plasmid 1981; 5:233-44. [PMID: 6115431 DOI: 10.1016/0147-619x(81)90001-9] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
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38
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Stougaard P, Molin S, Nordström K, Hansen FG. The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19. MOLECULAR & GENERAL GENETICS : MGG 1981; 181:116-22. [PMID: 6261081 DOI: 10.1007/bf00339014] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
The region of plasmid R 1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wild-type R 1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.
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39
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Nordheim A, Hashimoto-Gotoh T, Timmis KN. Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid. J Bacteriol 1980; 144:923-32. [PMID: 6254952 PMCID: PMC294754 DOI: 10.1128/jb.144.3.923-932.1980] [Citation(s) in RCA: 38] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.
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40
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Koekman BP, Hooykaas PJ, Schilperoort RA. Localization of the replication control region on the physical map of the octopine Ti plasmid. Plasmid 1980; 4:184-95. [PMID: 6100931 DOI: 10.1016/0147-619x(80)90008-6] [Citation(s) in RCA: 36] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
A series of small plasmids has been derived from the octopine plasmid pTi-B6 by in vitro manipulation. The smallest plasmid that is able to replicate in Agrobacterium tumefaciens contains the ampicillin-resistance determinant from Tn1, coupled to a piece of DNA that is homologous to HpaI fragment number 11 of the octopine Ti plasmid pTi-Ach 5. The incompatibility functions are also specified by this region.
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41
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Hooykaas PJ, den Dulk-Ras H, Ooms G, Schilperoort RA. Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens. J Bacteriol 1980; 143:1295-306. [PMID: 7410319 PMCID: PMC294500 DOI: 10.1128/jb.143.3.1295-1306.1980] [Citation(s) in RCA: 66] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid.
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42
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Molin S, Diaz R, Uhlin BE, Nordström K. Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop. J Bacteriol 1980; 143:1046-8. [PMID: 7009544 PMCID: PMC294415 DOI: 10.1128/jb.143.2.1046-1048.1980] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The replication control functions of a mutant of plasmid R1 that replicates without control at temperatures above 35 degrees C have been analyzed. Although the mutations have not been mapped precisely, the data indicate that the gene (cop) previously identified on the wild-type plasmid (S. Molin and K. Nordström, J. Bacteriol. 141:111-120, 1980) as being responsible for expressing a trans-acting replication inhibitor, as well as for incompatibility of plasmid R1, is not affected in this mutant. Thus, the conditional lack of replication control observed in this plasmid mutant presumably is not caused by the loss of inhibitor activity of the cop gene.
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43
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Bremer E, Beck E, Hindennach I, Sonntag I, Henning U. Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12: localization on hybrid plasmid pTU100 and expression of a fragment of the gene. MOLECULAR & GENERAL GENETICS : MGG 1980; 179:13-20. [PMID: 6256604 DOI: 10.1007/bf00268440] [Citation(s) in RCA: 60] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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44
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Miller CA, Cohen SN. F plasmid provides a function that promotes recA-independent site-specific fusions of pSC101 replicon. Nature 1980; 285:577-9. [PMID: 6250040 DOI: 10.1038/285577a0] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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