A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.     

High incidence of transposon Tn3 insertions into a replication control gene of the chimeric R/Ent plasmid pCG86 of Escherichia coli.

Insertion of transposon Tn3 into the chimeric R/Ent plasmid pCG86 occurred preferentially into a replication control gene, generating mutants with increased plasmid copy number. In two mutants, the nucleotide sequence of the region of the gene containing the Tn3 insertion was determined.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Plasmid pAS8Tc^s rep-1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas. The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.     

Restriction map of Tn7

Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7Δ6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.     

Characterization of the drug resistance plasmid NTP16

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.     

An inverted repeat sequence of the IncFI plasmid ColV2-K94 increases multimerization-mediated plasmid instability

A detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325. Inserts carrying the 1.2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance. One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell. A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure. This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host. These results suggest that the inverted repeat sequence of the X1 structure serves as a 'hot-spot' for generalized recombination. Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.     

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Selection and characterization of ColE1 plasmid mutants that exhibit altered stability and replication.

This report describes a method for isolating mutants of plasmid ColE1 that exhibit unstable maintenance and altered replication characteristics. It also describes the initial characterization of four mutants isolated by that method. A chimeric plasmid, pHSG124, containing a ColE1 derivative and a temperature-sensitive replication derivative of pSC101 was mutagenized in vitro, using hydroxylamine. By adjusting the growth conditions of transformants containing the mutagenized chimeric deoxyribonucleic acid, it was possible to rapidly screen colonies and identify those that had a high probability of carrying ColE1 mutants that exhibit unstable maintenance. Of those mutants, some exhibited altered copy number or accumulated catenated structures. Evidence is presented which suggests that the mutations in three of the mutants are probably located in the HaeII A fragment of ColE1.     

Control of F plasmid replication by a host gene: evidence for interaction of the mafA gene product of Escherichia coli with the mini-F incC region.

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.     

Overproduction of the Tn3 transposition protein and its role in DNA transposition

Five single base pair mutations that increase expression of the tnpA (transposase) gene of the Tn3 transposon approximately 30-fold, but which still allow the gene to be regulated, have been isolated by using a generally applicable procedure that involves distally linked lac gene fusions. The mutations, which are all located in a region controlling initiation of translation of the tnpA gene, do not affect normal repression of tnpA by the tnpR gene product, and yield up to a 9000-fold increase in tnpA protein production when combined with a tnpR mutation and placed on a high copy number plasmid. The mutation yielding the highest expression level was separated from the fused lac gene segment by homologous recombination and was found to increase the rate of transposition without altering the nature of the transposition product; in cells defective in both the E. coli recA gene and the tnpR gene of tn3, cointegrate transposition-intermediate structures occur with the overproducing--as well as with the wild-type--tnpA gene. In the presence of a functional Tn3 tnpR gene or the related transposon delta gamma, such cointegrate structures are resolved into the final products of transposition.     

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.