Transposition of a tetracycline-resistance determinant (Tn1523) and cointegration events mediated by the pIP231 plasmid in Escherichia coli

A 10.8-kb transposable DNA sequence conferring resistance to tetracycline resides on the IncY Escherichia coli plasmid pIP231. This sequence, designated Tn1523, was shown to insert into different sites of the replicons of the IncY prophage P1Cm c1.100 and the IncI1 plasmid pIP112. This process is not dependent on the host recombination system recA. Genetic results indicate that Tn1523 transposition involves the formation of a cointegrate intermediate, either between pIP231 and P1Cm c1.100, or between pIP231 and pIP112. These intermediates were found to be resolved into donor and recipient plasmids, each harboring a copy of the Tn1523 transposon. A stable structure formed by fusion of the pIP231 plasmid with the pIP112 plasmid was also observed. This event occurs in the absence of the bacterial recA gene product and seems to involve a site-specific reciprocal recombination between "IS-like" elements.     

Mutagenesis of dimeric plasmids by the transposon gamma delta (Tn1000).

The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon gamma delta (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single gamma delta insertion, the occasional recovery of transposon-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization. We show here that gamma delta-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec+.     

Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.     

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.     

Intermolecular Transposition of Is10 Causes Coupled Homologous Recombination at the Transposition Site

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 P(R) promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in δrecA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.     

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.     

Replication Control and Switch-Off Function as Observed with a Mini-F Factor Plasmid

Mini-F is a fragment of the F plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. Its copy number is one to two per chromosome. This plasmid is joined to ColE1, whose copy number is 16 to 20. Under normal circumstances the composite plasmid replication exhibited ColE1 characteristics, maintaining a high copy number. However, when ColE1 replication was inhibited by deoxyribonucleic acid polymerase I inactivation, its replication exhibited mini-F characteristics, maintaining a low copy number. These observations are in complete agreement with those of Timmis et al. (Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560, 1974), who examined the behavior of a recombinant plasmid formed between pSC101 and ColE1. The transition from high to low copy number allowed us to examine the control system acting in cells carrying plasmids exhibiting intermediate copy numbers. The initiation of the mini-F replication system as represented by deoxyribonucleic acid synthesis of the composite plasmid was completely blocked when there were multiple copies of mini-F in a cell. It was not restored until the copy number was lowered to one to two, after which replication was first detected. ppF, a mini-F replicon packaged in a phage λ head behaved similarly: its replication was completely shut off when the resident mini-F genome copy number was high and was inhibited partially when the resident mini-F genome copy number was low. These experiments clearly demonstrate that there is a switch-off mechanism acting on deoxyribonucleic acid synthesis (initiation) in a cell carrying mini-F, and its intensity is related to the plasmid copy number. This result supports the “inhibitor dilution model” proposed by Pritchard et al. (Symp. Soc. Gen. Microbiol. 19:263-297, 1969). The nature of the hypothetical inhibitor is discussed.     

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.     

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.     

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.     

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.     

Tn 7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.     

Plasmids for recombination-based screening.

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.     

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

Conjugal mobility of the multicopy plasmids NTP1 and NTP16

Mobilization of the multicopy plasmid NTP16, like that of ColE1, is promoted by a range of conjugal plasmids. However, the mechanisms employed for NTP16 mobilization differ between groups. Mobilization by the IncI1 plasmid R64 requires trans-acting products from NTP16 plus a cis-acting region of the small plasmid. In contrast, this system is used inefficiently by the F plasmid and instead, a high-frequency conduction process occurs. Analysis of exconjugant cells reveals that F-mediated mobilization of NTP16 frequently involves rearrangements of NTP16 DNA, promoted by the Tn1000 transposon of F and/or by the kanamycin resistance transposon (Tn4352) of NTP16. Possible mechanisms for the high-frequency F-mediated mobilization of NTP16 are discussed. The plasmid NTP1, which is closely related to NTP16, is also mobilized efficiently by R64. It is not however efficiently mobilized by F, demonstrating the requirement for the Tn4352 element, which is not present in this plasmid, for effective F-mediated transfer.     

RecA independent,site-specific recombination between ColE1 or ColK and a miniplasmid they complement for mobilization and relaxation: Implications for the mechanism of DNA transfer during mobilization

We report a novel type of recA independent recombination between plasmids ColE1 or ColK and a naturally occurring miniplasmid (pLG500). This miniplasmid can be complemented for mobilization and relaxation in the presence of ColE1 or ColK. Recombination between ColE1 and pLG500, or ColK and pLG500, was site-specific, and was only detected following the mobilization of these plasmids. The composite plasmids thus formed were stable, but recombination (resulting in dissociation of their component replicons) was again detected following mobilization. For ColE1, the site at which cointegration with pLG500 occurred was mapped to within 47 base pairs of the relaxation nicking site; for ColK, the recombination site was localized to the same region as its genetically defined transfer origin. The generation of these cointegrate plasmids is consistent with the hypothesis that mobilization entails relaxation nicking, transfer of the nicked single strand of DNA, and recircularization of the transferred single strand by ligation of 3′ and 5′ termini by the relaxation protein bound to the 5′ nick terminus. Since both plasmids are mobilized by the same proteins, their cointegration can be explained as a consequence of the ligation of the 5′ end of one plasmid to the 3′ end of the other, and vice versa.