The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility

The incompatibitity 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 superheliclty, 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 enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Abnormal Reaction Product from Condensation of Phloroaceto-phenone Dimethylether with p-Methoxybenzaldehyde

We identified and analyzed a DNA region that is required for the stable maintenance of plasmids in the genus Sphingomonas. This DNA fragment, a 244?bp, is localized in the upstream region of the repA gene of low-copy-number small plasmid pYAN-1 (4896?bp) of Sphingobium yanoikuyae. It has four inverted repeats and one direct repeat for possible secondary structures. We were able to stabilize not only another unstable plasmid, pYAN-2, in the genus Sphingomonas, but also the unstable plasmid pSC101 without par locus in Escherichia coli. The copy-number levels between the unstable plasmid and the parental plasmid were similar, and these results suggest that the stabilization of unstable plasmids by this DNA region of pYAN-1 was not due to an increase in plasmid copy number. We concluded that the stabilization of the plasmid was due to a plasmid partition mechanism encoded by a DNA fragment of pYAN-1.     

Isolation of a Derivative ofEscherichia coli–Enterococcus faecalisShuttle Vector pAM401 Temperature Sensitive for Maintenance inE. faecalisand Its Use in Evaluating the Mechanism of pAD1par-Dependent Plasmid Stabilization

A derivative of theEscherichia coli–Enterococcus faecalisshuttle vector pAM401 was isolated by mutagenesis in anE. colimutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38°C than at 30°C in theE. faecalishost strain JH2-2. TheE. faecalisplasmid pAD1-encodedparstability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found thatparstabilized pAM401ts at 38°C but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38°C. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putativeparantidote,in transattenuated cell killing. These characteristics support a postsegregational killing mechanism forpar-mediated plasmid stabilization.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region

Summary The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of -lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that 1.9x10⁶ daltons of the 6.0x10⁶ dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P_vgb of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P_vgb was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P_vgb provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P_vgb, while a construct containing P_vgb, the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P_vgb, vgb operon) or pKVpPV (P_vgb, vgb operon, par) slightly attenuated (30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P_vgb, egfp) or pKVpP-E (P_vgb, par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P_vgb, vgb operon, par, tod) or pKVpP-T (P_vgb, par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.     

Partitioning of plasmid R1 in Escherichia coli : II. Incompatibility properties of the partitioning system

A theoretical as well as an experimental study of the effect of the partitioning system on plasmid R1drd-19 incompatibility was performed. The theoretical numerical analysis (by computer) was based upon the following assumptions: (i) The partitioning (par) mechanism is independent of the replication (rep) and replication control (cop) mechanism. (ii) A par mutation causes random (binomial) distribution of plasmid copies between the daughter cells at cell division. (iii) In the par⁺ case, the plasmid copies are equipartitioned and selected randomly for partitioning. (iv) Selection of plasmid copies for replication is random. (v) Two different replication control systems were considered: Model 1 assumes that the plasmid copy number is set to exactly 2n before cell division, whereas in Model 2 exactly n copies are synthesized per cell per cell cycle. Numerical analysis was performed for the n values 2–8. The result was that in all cases (par⁺/par⁺, par⁺/par, par/par), steady states with respect to copy number distribution within the heteroplasmid population were rapidly (within five or six generations) established, giving constant loss rates. The rate of loss was slightly higher in the par/par case than in the other two. The two replication control models gave almost identical loss rates. In the par⁺/par case, the par⁺ plasmid had an advantage over the par plasmid. The experimental approach was to create heteroplasmid populations of Escherichia coli by introducing two genetically marked derivatives of plasmid R1drd-19 and then follow the reduction in the relative size of this population during exponential growth in LB medium. The loss rate was essentially the same in the par⁺/par⁺ and par⁺/par combination and slightly higher in the par/par case, suggesting that plasmid incompatibility mainly is caused by the replication and copy number control system. In the par⁺/par situation, the par⁺ plasmid had a pronounced advantage over the par plasmid. The par region of plasmid R1 (without the basic replicon) was cloned onto the vector pSF2124. A par (deletion) mutation was not complemented by par⁺. Plasmid pSF2124, which does not seem to carry a par system of its own, could use the R1 par system, adding to the conclusion that par is independent of rep and cop. Plasmids pSF2124 and R1drd-19 are completely compatible, whereas plasmid pSF2124 carrying the R1 par system and plasmid R1drd-19 showed a weak incompatibility although the copy numbers of the two plasmids were not affected in the heteroplasmid cells. Hence, the partitioning system causes incompatibility, but the effect is weak compared to that of the cop system. The result is consistent with some sort of assortment of plasmids before partitioning.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus.

Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene. While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability. Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding. Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions. They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.     

Organization of chimeras between filamentous bacteriophage f1 and plasmid pSC101

Two recombinants formed in vivo between the filamentous phage f1 and the tetracycline-resistance-conferring plasmid pSC101 are capable of transducing sensitive cells to Tet^r. These chimeric filamentous phage, VO-1 and VO-2, were previously shown to contain the entire f1 and pSC101 genomes (Vovis et al., 1977; Ohsumi et al., 1978). The genomes of VO-1 and VO-2 are unstable in vivo; VO-1 breaks down to yield a molecule similar to pSC101 and an f1-like species, f1′. f1′ was previously shown to differ from f1 by the presence of 209 additional nucleotides inserted in the carboxy-terminal portion of gene IV (Ravetch et al., 1979). We have found by hybridization analysis and direct DNA sequencing that this 209-nucleotide segment is present in one copy in pSC101, and that it has properties similar to known transposable elements. Therefore, we have called this sequence IS101. We have characterized the structures of both VO-1 and VO-2 in greater detail by restriction mapping and DNA sequence analysis. Both chimeras contain two copies of IS101, which are present as direct repeats and form the junctions between the f1 and pSC101 genomes. The IS101 elements in VO-1 and VO-2 are flanked by a five-base direct repeat of f1 sequence that is not repeated in wild-type f1. The junction between f1 and pSC101 in VO-1 is located at the same point as the IS101 element in f1′, while in VO-2 the junction between the two genomes is at a point in f1 located between the promoter and ribosome binding site for gene VIII. The pSC101-like molecules derived from the breakdown of VO-1 in vivo are identical to the original pSC101 in the region of IS101. The IS101 elements in the original and derived pSC101 plasmids are not flanked by any repeated sequence. Attempts to regenerate VO-1 from f1′ and pSC101, both of which contain one IS101 element, indicate that the breakdown of VO-1 is irreversible. These results are discussed in terms of current models for transposition, which postulate structures similar to VO-1 and VO-2 as intermediates in transposition.     

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the λ p_R promoter, the c1857 temperature-sensitive represser gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of > 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.     

Role of DNA superhelicity in partitioning of the pSC101 plasmid

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.     

Genetic analysis of the inter-relationship between plasmid replication and incompatibility

Summary The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1.The inability of the composite plasmid to replicate at 42° in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nevertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.     

Altered tetracycline resistance in pSC101 recombinant plasmids

Summary Investigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing of EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance. The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).     

Stable maintenance in chemostat-grownEscherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene

Plasmid stability was studied in antibiotic-free chemo-stat cultures . Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.Issued as NRCC publication No. 23992.     

Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts

Summary We cloned the pem segment of plasmid R100 containing the two genes pemI and pemK, which are responsible for stable maintenance of R100 in dividing cells, into pHS1, a temperature-sensitive replication mutant of plasmid pSC101. We then examined the effect of the pem system on the maintenance of the resultant pem ⁺ plasmid pDOM17 in various Escherichia coli host strains upon inhibition of replication of the plasmid at a high temperature. We show that the pem ⁺ plasmid was maintained stably in the cell population and efficiently in the two hosts, km1213 (polA ^ts) and KP64 (recA), but less efficiently in others, such as W3110, C600, P3478 (polA), and SH2743 (sfiA sfiC); the rate of cell growth was reduced at or after the time when the copy number of pDOM17 was supposed to be 0 in all of the hosts examined. We also show that a large fraction of the non-viable pDOM17-free segregant cells was produced in the former two hosts, while a smaller fraction of such cells was produced in the latter hosts, in which cell division was inhibited for several generations. Based on these results and other observations, we point out that the pemK gene product has the function not to kill the plasmid-free segregant cells, but primarily to inhibit division of these segregants. Inhibition of cell division secondarily leads to death of the plasmid-free segregants very efficiently in the two particular hosts, resulting in an apparently more stable maintenance of the pem ⁺ plasmid in these two hosts than in others.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC.

Summary The rate of replication of the plasmids colE1, pSC101, R100.1 and pAR132 (an RTF-TC derivative of the drug resistance factor R100.1) has been investigated directly by DNA: DNA hybridization. These rates have been compared, in a dnaAts strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures. Chromosome initiation in the dnaAts strain stops rapidly after a shift to the non-permissive temperature, but plasmids R100.1 and pAR132 do not seem to be affected directly and continue replication for some time. The colE1 replication rate undergoes a large increase after the temperature shift, followed by a rapid decrease to a very low level 25 min after the shift. In contrast pSC101 replication stops immediately after the shift. ColE1 is able to replicate in an integratively suppressed dnaAts strain at 42° C whereas pSC101 stops replication immediately under these conditions. We conclude that R100.1 and its derivative RTF-TC can replicate without a functional dnaA product; that colE1, while affected by a shift in temperature in a dnaAts strain, does not directly require dnaA; and that the plasmid pSC101 has an absolute requirement for dnaA. The absolute requirement of pSC101 for dnaA in the integratively suppressed Hfr strain provides a useful system for further investigations of the dnaA function.