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1.
We have constructed a mini-F derivative (pKP1013) consisting of a 5.4 kilobase pairs (kb) segment (44.0 to 49.4 kb) of mini-F and fragments carrying the chloramphenicol and spectinomycin resistance genes that originated from the R plasmid NR1. The plasmid pKP1013 replicates autonomously in a manner indistinguishable from that of the parental mini-F. An amber mutant defective in replication has been isolated from pKP1013 by localized mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The virtual absence of incorporation of [3H]-thymidine into the plasmid DNA as well as the kinetics of appearance of plasmid-free segregants suggest that plasmid DNA synthesis is primarily affected under nonpermissive conditions. The amber mutation has been mapped within the 530 base pairs (bp) region that extends from 45.25 (XmaI) to 45.78 Kb (PstI) by extensive analysis of in vitro recombinants constructed from rep+ and rep- plasmids.  相似文献   

2.
Summary Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accuratepartitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG+ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.  相似文献   

3.
Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.  相似文献   

4.
The par region of mini-F is both necessary and sufficient to promote equipartition of plasmid copies to daughter cells. It is approximately 2.5 kb long and contains the coding sequences for two proteins, F1 (41 kDa) and F2 (37 kDa). We isolated 13 mutants of a phage λ-mini-F hybrid that form unstable plasmids. Two of these putative Par? mutants are fully suppressible nonsense (amber) mutants. One of the amber mutants, par-41, eliminates the synthesis of F1, generating a large nonsense fragment of the protein. The other mutant, par-36, eliminates the synthesis of F2. Thus both proteins appear to be essential for plasmid equipartition.  相似文献   

5.
We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.  相似文献   

6.
Summary The hopE mutants of Escherichia coli, which cannot stably maintain a mini-F plasmid during cell division, have mutations in the recD gene coding for subunit D of the RecBCD enzyme (exonuclease V). A large amount of linear multimer DNA of mini-F and pBR322 plasmids accumulates in these hopE mutants. The linear multimers of plasmid DNA in the hopE (recD) mutants accumulate in sbc + genetic backgrounds and this depends on the recA + gene function. Linear plasmid multimers also accumulated in a recBC xthA triple mutant, but not an isogenic xthA mutant or an isogenic recBC mutant. The recBC xthA mutant is defective in the conjugative type of recombination. Linear plasmid multimers were not detected in the recBC strain. We propose models to account for linear multimer formation of plasmids in various mutants.  相似文献   

7.
The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.  相似文献   

8.
Chieko Wada  Takashi Yura 《Plasmid》1982,8(3):287-298
When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [3H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.  相似文献   

9.
H Uga  F Matsunaga    C Wada 《The EMBO journal》1999,18(13):3856-3867
In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.  相似文献   

10.
hopA mutants, which have been suggested to be defective in mini-F plasmid partitioning (H. Niki, C. Ichinose, T. Ogura, H. Mori, M. Morita, M. Hasegawa, N. Kusukawa, and S. Hiraga, J. Bacteriol. 170:5272-5278, 1988), were found to carry mutations in the gyrB gene, coding for the B subunit of DNA gyrase. In gyrB(HopA) mutants, relaxation of the superhelicity of plasmids, increased IncG incompatibility, and increased SopB protein production were observed. It is suggested that altered expression of the sop genes, which is due to relaxation of the mini-F plasmid DNA, causes both defective partitioning of the mini-F plasmids and increased IncG incompatibility in gyrB(HopA) mutants.  相似文献   

11.
The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.  相似文献   

12.
13.
Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.  相似文献   

14.
Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.  相似文献   

15.
Isolation of nonsense suppressor mutants in Pseudomonas.   总被引:31,自引:13,他引:18       下载免费PDF全文
A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.  相似文献   

16.
C Wada  T Yura 《Journal of bacteriology》1984,160(3):1130-1136
Replication of F (including mini-F) and some related plasmids is known to be specifically inhibited in mafA mutants of Escherichia coli K-12. We have now isolated and characterized mini-F mutants that can overcome the replication inhibition. Such plasmids, designated pom (permissive on maf), were obtained spontaneously or after mutagenesis with hydroxylamine or by transposon (Tn3) insertion. In addition to their ability to replicate in mafA mutant bacteria, the pom mutant plasmids exhibit an increased copy number and resistance to "curing" by acridine dye in the mafA+ host. In agreement with these results, Tn3-induced pom mutants were found to carry Tn3 inserted at the incC region of mini-F DNA, known to be involved in incompatibility, control of copy number, and sensitivity to acridine dye. Furthermore, three of the seven mini-F plasmids tested that carry Tn3 within the tandem repeat sequences of the incC region (previously isolated by other workers) exhibit all the phenotypes of pom plasmids, the ability to replicate in the mafA strain, and high copy number and acridine resistance in the mafA+ strain. The rest of the plasmids that contain Tn3 just outside the tandem repeats remain wild type in all these properties. These results strongly suggest that the putative mafA gene product of host bacteria controls mini-F replication through interaction with the incC region.  相似文献   

17.
Mutants have been constructed by deleting regions of the gene rpsA for ribosomal protein S1, which had been cloned in plasmid pACYC184. The mutant genes were analyzed for their ability to complement an S1 amber mutant containing a temperature-sensitive suppressor. Another series of mutants was constructed using the tac promoter plasmid pKK223-3, and the effect of the mutant proteins was analyzed in a strain wild type for rpsA. The gene products of all mutants were identified by the immunoblotting technique. Plasmids with a mutant rpsA gene which do not or only poorly complement the S1 amber mutation cause drastic growth reduction, whereas the overall protein synthesis is affected to different extents depending on the site of the deletion. Mutants which express S1 fragments comprising at least the NH2-terminal 100 amino acids stimulate or inhibit the synthesis of certain cellular proteins. The amount of chromosomal coded S1 was reduced by each mutant plasmid. Our data suggest that S1 has a general regulatory role during protein biosynthesis.  相似文献   

18.
The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.  相似文献   

19.
20.
E P Ogryz'ko  V G Nikiforov 《Genetika》1988,24(10):1894-1896
A multicopy plasmid pLMN1 expressing a wild type rpoB gene encoding Escherichia coli RNA polymerase beta subunit gene was constructed. Introduction of this plasmid into rifampicin-resistant RpoB mutants makes them rifampicin-sensitive. Rifampicin-resistant clones appear in such strains with frequencies up to 10(-3), due to recombinational (recA-dependent) transfer of rif-r mutations from chromosome to pLMN1. This provides a simple selection procedure for transfer of any rpoB mutation, together with a rif-r mutation from a chromosome to pLMN1. In this way, we transferred rpoB22 amber mutation to pLMN1 for localization of the mutant codon by DNA sequencing.  相似文献   

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