Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Differential effect of hydroxyurea on the replication of plasmid and chromosomal DNA in Bacillus subtilis.

The replication in Bacillus subtilis of the staphylococcal R plasmids pE194, pBD15, pUB110, pSA0501, and pSA2100 has been studied in the presence of hydroxyurea. In all cases, an enrichment for covalently closed circular DNA compared with chromosomal DNA was observed. In this respect, hydroxyurea mimics the effect previously observed with pUB110, using strains carrying the conditional mutation dnaA13. This mutation has been reported to affect ribonucleotide reductase (G. W. Bazill and D. Karamata, Mol. Gen. Genet. 117:19-29, 1972). An explanation for these effects is offered, together with some supporting evidence.     

On the identity of dnaP and dnaF genes of Bacillus subtilis

Summary The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.Abbreviations and symbols HPUra 6-(p-hydroxyphenylazo)-uracil; mic, minimum inhibitory concentration - MMS methyl-methanesufonate - Pol I Pol II and Pol III: DNA polymerase I, II and III respectively - PCMB parachloro-mercuri-benzoate     

Functional analysis of the dna (Ts) mutants of Bacillus subtilis: plasmid pUB110 replication as a model system

Summary We determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication. Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III. When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III. In oriL ^- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other. The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32. At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis.     

Transformation and transduction of Bacillus subtilis strains with the Bsu R restriction-modification system by means of modified and unmodified DNA of pUB110 plasmid

Summary During transformation of B. subtilis cells with the Bsu R restriction-modification system by means of pUB110 plasmid restriction and modification of the plasmid DNA occurs. The effect of restriction on the transformation frequency is relatively weak, bringing about 20-fold decrease only. When using cells of a modifying recipient, the frequency of AR9 phage-mediated transduction of unmodified plasmid DNA is also relatively little decreased. The frequency of transduction by chromosomal markers, under the same conditions, falls much lower.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

A phenomenon of plasmid retrotransfer in conjugation of Bacillus subtilis

A conjugative retrotransfer-retromobilization of a small nonconjugative plasmid pUB110 was established in Bacillus subtilis. This process involves a large conjugative plasmid p19.     

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.     

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Complementation of a dnaC initiation defect in vitro

Summary The dnaC28 mutant, CT28-3b, is an initiation defective dnaC strain. Extracts of the mutant failed to synthesize DNA in vitro when the strain was incubated at the restrictive temperature for two generation times prior to preparation of the extract. Addition of a complementing extract from a Col-E1::dnaC ⁺ hybrid plasmid containing strain or of partially purified dnaC protein resulted in substantial synthesis. Hybridization of the DNA made by these in vitro complementation extracts showed that a significant portion of this DNA was from the region near the chromosomal origin of replication.     

Transfer of pUB110 plasmid via spontaneous transformation in Bacillus subtilis

During the growth of Bacillus subtilis strains in mixed culture, the transfer of the plasmid pUB110 from cell to cell occurs via spontaneous transformation.     

A Useful Cloning Vector for Bacillus subtilis

We have constructed plasmid pDN1050 a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells.

The minicell system of Bacillus subtilis has been used to study the expression of plasmid genes using several R plasmids derived from Staphylococcus aureus. pE194, pC194, and pUB110 as well as several mutant and in vitro recombinant derivatives of these plasmids segregate into minicells. A copy control mutant of pE194 was used to show that the extent of segregation is proportional to the copy number. The polypeptides specified by these plasmids were examined by SDS-polyacrylamide gel electrophoresis. Six proteins specified by pE194, an erythromycin resistance plasmid, were identified using cop mutants. These comprise about 90% of the potential coding capacity of the 2.4-Mdal pE194 plasmid. One of these proteins (29,000 daltons) is inducible by erythromycin in the wild type pE194 but is synthesized constitutively in a mutant derivative which also expresses antibiotic resistance constitutively. Several other proteins are detected only in copy control mutants. pUB110, a kanamycin resistance plasmid, expresses three major proteins which comprise 50% of the coding capacity of this 3.0-Mdal plasmid. Two additional minor proteins are occasionally observed. pC194 (2.0 Mdal), which confers chloramphenicol resistance, expresses two polypeptides comprising about 25% of its coding capacity. One of these polypeptides (22,000 daltons) is inducible by chloramphenicol. pBD9, an in vitro composite of pUB110 and pE194, probably expresses all of the major parental plasmid proteins with the exception of one from pUB110 and one from pE194.     

Use of bacilli for overproduction of exocellular endo-β-1,4-glucanase encoded by cloned gene

A Bacillus subtilis endo-β-1,4-glucanase gene contained in a recombinant plasmid pBS1 was transferred into a new shuttle vector plasmid pCK98 by ligating linearized DNAs of pBS1 and pUB110. B. subtilis RM125 and B. megaterium transformed with pCK98 produced the glucanase substantially and excreted into the medium. Most of the enzyme was produced during the exponential growth period and the production was not repressed by glucose or cellobiose. The plasmid was stable in B. megaterium but not in B. subtilis.     

Origin and mode of replication of plasmids pE194 and pUB110

Replication of the multicopy antibiotic resistance plasmids pE194 and pUB110 has been studied in Bacillus subtilis. Based on electron microscopic analysis of replication intermediates and on evidence from deletants, we have determined that both plasmids replicate unidirectionally from fixed origins by a Θ mechanism. The location of these origins and the directions of replication have been determined relative to the physical maps. A cointegrate of pE194 and pUB110 has also been studied and evidence is presented from electron microscopy, temperature resistance, and copy number analysis that the cointegrate uses both parental replication origins.     

Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates.

Summary The role of E. coli dnaB and dnaC protein in the replication of plasmid ColE1 and RSF1030 DNA was investigated in a soluble in vitro system (Staudenbauer, 1976a). Extracts from dnaB and dnaC mutants which are phenotypically DNA initiationor DNA elongation-defective were examined for their replicative capacity. It was found that all mutants tested are deficient in the synthesis of supercoiled plasmid DNA. Deficient extracts of dnaB mutants could be partially complemented by purified dnaB wild type protein but required for full complementation dnaC wild type protein as well. The dnaB wild type protein could be replaced by a P1dnaB analog (ban) protein complexed with a dnaB ts protein. Deficient extracts of dnaC mutants were complemented by purified dnaC wild type protein alone.The in vitro plasmid replication cycle had been separated into an early and late stage (Staudenbauer, 1977). Analysis by CsCl velocity centrifugation of the plasmid DNA synthesized in mutant extracts indicates that the early stage, namely the synthesis of early replicative intermediates, proceeds in all dnaB and dnaC mutants tested. However, replication of the early intermediates during the late stage depends on both the dnaB and dnaC protein. These conclusions were confirmed using inhibitors of DNA synthesis.     

A functional map of the replicator region of the octopine Ti plasmid

A hybrid plasmid of pUB 110 (Neo^r) and pAB 124 (Tc^r) has been constructed and shown to have a Neo^sTc^r phenotype in Bacillus subtilis. A derivative of this pUB 110:pAB 124 hybrid has been isolated, pAB 324, which has the expected Neo^rTc^r phenotype. A restriction endonuclease cleavage map of pAB 324 was compared to that of the parent hybrid. This showed that pAB 324 contained a minimum of two deletions and one insertion. This insertion (approximately 1.0 Md) has been identified as originating from the Bacillus subtilis chromosome.     

Protein synthesis induced by infection with packaged lambda dv plasmid

Plasmid λdv or imm²¹dv DNA was joined to a λ arm having a cos site. This recombinant plasmid can be packaged in a λ head, and used to infect Escherichia coli K12 cells. The injected DNA molecules become plasmids in cells. By adding these particles to uv-irradiated uvrA cells, the packageable λdv or imm²¹dv plasmids can be induced to synthesize proteins coded by genes on the plasmid genome. The packageable plasmid system is thus suitable for studying on synthesis and regulation of plasmid-coded biopolymers. Analyses of the dv-coded proteins in gel electrophoreses revealed that among several genes carried on the dv plasmid genome, only those genes that are members of the pRoR-tof-cII-O-P operon can be expressed. Evidence has been presented to show that expression of this operon, which is directly correlated with replication of the genome, is only partially allowed in cells perpetuating the dv plasmid. These observations are discussed in connection with the autorepressor model (D. E. Berg, 1974, Virology62, 224–233; K. Matsubara, 1976, J. Mol. Biol.102, 427–439) that genetically accounts for the control mechanism of plasmid replication.