Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli

We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (O^c) mutations on the pBR322-derived plasmid pPY97. The rate of O^c mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the O^c phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Participation of the uvrD gene in the precise excision of the Tn9 transposon

HfeTn5-(04,06) and IfeTn9 mutations increase efficiency of precise excision of Tn5, Tn10 and decrease that of Tn9. These mutations have been mapped in uvrD gene. In LFETn9 and UVRE502 mutants, the multicopy plasmid pEM61 carrying the cloned uvrD gene complements LFETn9- phenotype (Low Frequency Excision of Tn9). These results indicate that the uvrD gene product plays different role in excision of transposons with long and short inverted repeats. The mechanism of this effect is discussed.     

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.     

Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli

The transposon Tn5 contains a unique central region bordered by 1.5-kb inverted repeats. The in vitro deletion of the centre of Tn5, with a restriction endonuclease (XhoI) which cuts within the inverted repeats leads to the production of a palindrome on subsequent ligation. This palindromic region is unstable on subsequent transformation into Escherichia coli (Collins, 1981). Precise excision of the Tn5 region plus one copy of the bracketing 9-bp direct repeat occurred in about one-third of the transformants. The rest of the transformants contain only remnants of the inverted repeat. Sequence analysis indicated that deletion had occurred between short direct repeats. The precise excision of these "nearly precise" excision products continued with high frequency and was found to be affected by mutations that interfere with the normal precise excision of transposons. In a recB, sbcB host precise excision was markedly reduced. A common mechanism is proposed for all recA-independent deletions occurring in E. coli.     

Fluctuation tests are more sensitive than plate tests in detection of chemicals which cause enhanced excision of transposon Tn10 in Salmonella typhimurium

Precise excision of transposon Tn10, as judged by reversion of Salmonella typhimurium strain LT2 trp1014::Tn10 to Trp+, was not detectably enhanced following exposure to 9-aminoacridine, 5-azacytidine or mitomycin C in conventional treat-and-plate assays. By contrast, 7/13 chemicals, including 5-azacytidine and mitomycin C, were found to be capable of enhancing precise excision of Tn10 when tested in modified fluctuation assays. Despite earlier reports, precise excision is one activity of transposons which is not therefore refractory to enhancement by chemical mutagens.     

Isolation of Escherichia coli K12 mutants with deficient in precise excision of transposons

Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid. The frequency of precise excision of Tn5 from plasmidic genome is 10(-5). The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision. Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders. The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.     

RecA-dependent increased precise excision of Tn10 in Salmonella typhimurium.

UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella typhimurium strain; mitomycin C was also found to induce the frequency of precise excision of Tn10 from srl or met. Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from a uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10. In contrast to point mutations excision of Tn10 was found to be UV inducible in a top mutant.     

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.     

Characteristics of Escherichia coli K-12 mutants with altered effectiveness of excision of Tn5 and Tn9 transposons

The properties of Escherichia coli K-12 mutans HFETn5, HFETn9 and LFETn9 have been studied. The majority of mutations were shown to have pleiotropic effect. Some of them increase cell sensitivity to UV light and mitomycin C and affect efficiency of homologous recombination in transduction and conjugation. The level of spontaneous mutagenesis is increased in a number of mutants. None of the mutations isolated affect frequency of transposition of Tn5 from bacteriophage lambda::Tn5 into the chromosome. Based on analysis of properties of hfeTn5-09 and hfeTn9 mutations and on the date of preliminary mapping of hfeTn5-09 mutation, these mutations were considered to be novel. It is shown that the processes of precise excision of Tn5 and Tn9 transposons may be accomplished by at least two pathways, one of them being dependent on recA gene functions.     

New class of mutations in Escherichia coli (uup) that affect precise excision of insertion elements and bacteriophage Mu growth

We have used a papillation screening technique to isolate mutations that increase the precise excision of insertion elements. The three mutations isolated stimulated precise excision of Tn5, Tn10, and the IS elements. They had a large, 20- to 600-fold, effect on excision of Tn5 at various chromosomal sites. The varied stimulation for different Tn5 insertions showed that the mutations altered the relationship between a precise excision activity and the chromosomal sequence flanking an inserted Tn5. A much smaller stimulation was observed for insertions on the plasmid F'128. The stimulation was recA independent. The mutations also reduced the rate of production of bacteriophage Mu progeny. The mutations were mapped by two- and three-factor crosses with closely linked Tn10 insertions. They defined the uup locus, located at 21.3 min on the Escherichia coli map, next to pyrD.     

Effect of dioxidine, antineoplastic agents and other mutagens on the precise excision of Tn1 and Tn10 transposons in E. coli K12

Induction of precise excision of transposons Tn1 and Tn10 from the genes met::Tn1 and cys::Tn10 by chemical agents, having mutagenic and DNA damaging activities, has been studied. The drugs dioxydin, NMU, photrin, phopurine, thiophosphamid, rongeron as well as sodium azide, 2-NP, DDDTDP are shown to differ in their ability to stimulate the precise excision of transposons of different classes and in the efficiency of stimulated process. Results of the present paper are in proof of the possible using of experimental model, based on registering the precise excision of transposons, for screening the mutagenic and cancerogenic activities of chemical agents from the environment.     

Insertion of a transposon for chloramphenicol resistance into bacteriophage Mu.

We have isolated mutants of bacteriophage Mu carrying the X mutations caused by the insertion of cam (Tn9), a transposon for chloramphenicol resistance. The Mu X cam mutants were obtained by selecting for heat-resistant survivors of a Mucts62, P1cam dilysogen. Like the previously described X mutants, Mu X cam mutants are defective prophages which can be excised from the host DNA at a frequency of 10(-5) to 10(-7) per cell. Tn9 insertions in Mu X cam mutants are located within 5000 base pairs of the left end of Mu DNA in a region that controls early replication functions of Mu. There is one EcoRI cleavage site in Tn9. The Tn9 transposon itself can be excised precisely from the Mu X cam mutants to generate wild type Mu. In most Mu X cam mutants, precise excision of Tn9 occurs at a low frequency (10(-6) per cell), whereas in some, the frequency is higher (10(-4) per cell). Mu X cam prophages can replicate after induction with the help of wild type Mu. The lysates containing Mu X cam particles, however, fail to transduce chloramphenicol resistance at a high frequency; Mu X cam mutants apparently have a cis dominant defect in integration.     

Antimutagenic effects of chemicals on mutagenesis resulting from excision of a transposon in Salmonella typhimurium

We have found that a temperature-sensitive mutation in the polA gene of Salmonella typhimurium strain LT2 causes precise excision of transposon Tn10 to occur at significantly increased frequencies in cells incubated at the restrictive temperature. In our experiments, precise excision from a site in the tryptophan operon was measured by determining the frequency of reversion of the auxotrophic trp1014::Tn10 polA7 strain to prototrophy on defined medium containing a trace amount of broth. Because the yields of revertants at 37 degrees C were of the order of 200 colonies per plate, it was possible to measure the effects of chemical inhibitors on the processes involved in precise excision. We now report that all of the DNA-repair inhibitors we have studied (caffeine, ethionine, acriflavine, procaine and cinnamaldehyde) are effective inhibitors of precise excision of Tn10, and can therefore be defined as antimutagens.     

Modulation of mutagenesis involving precise excision of transposon Tn10

Precise excision of transposon Tn10 results in reversion of the Trp- phenotype to Trp+ in a trp-1014::Tn10 strain of Salmonella typhimurium, and also occurs at a markedly higher frequency in a strain carrying the temperature-sensitive polA7 allele. The frequency with which precise excision events occurs can be modified by the plating medium, results indicating that the great majority of mutants which arise on broth-supplemented or tryptophan-supplemented minimal media actually arise on the selective plating medium. Trp+ revertants (1000) arising from excision of Tn10 were purified by re-streaking for single colonies; none were found to retain the Tn10 encoded resistance to tetracycline. Yields of Trp+ revertants of the polA7 strain were consistently higher when glycerol rather than glucose was used as sole carbon source in the selective medium. Clean excision of Tn10 can also be increased by ultraviolet irradiation in (R) plasmid-free strains, and is further increased in strains carrying an N-group plasmid (R205, R46 or pKM101). Ultraviolet-induced precise excision of Tn10 also occurs at a much enhanced frequency in a strain with a deletion through the uvrB gene; in this case, however, the addition of plasmid pKM101 leads to a decrease in yields of ultraviolet-induced precise excision events.     

Genetic evidence for absence of transposition functions from the internal part of Tn981 a relative of Tn9

Summary Inverse transposition of the DNA of pBR322 was found to be mediated by the small transposon Tn981 a relative of Tn9 flanked by direct repeats of IS1. Since the resulting structure IS1:: pBR322::IS1 (Tn983) is transposed in a second step in the absence of Tn981, it is concluded that all the functions necessary for transposition of IS1 flanked transposons are coded for by IS1 itself or the E. coli chromosome, respectively.     

Precise excision of transposons and point mutations induced by chemicals.

The ability of 23 chemicals (carcinogens and non-carcinogens) to induce precise excision of Tn10 and point mutations was studied in experiments with a single strain. The mutation assay was shown to detect a wider spectrum of genotoxic agents than the assay of Tn10 precise excision. The latter was induced only by potent SOS mutagens, which is in accordance with data on the SOS dependence of the induction of precise excision of Tn10. The precise excision assay as an additional test contributing to the knowledge of particular features of the action of a tested mutagen is discussed. The induction of precise excision of Tn10 by pyrene (and its failure to induce point mutations in this strain) demonstrates the value of using the transposon excision assay in cases of 'problem' mutagens.     

Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.     

Participation of DNA polymerase II in the increased precise excision of Tn10

In this work the involvement of polymerase II (Pol II) in the precise excision of Tn10 stimulated by a dnaB252 thermosensitive (Ts) mutant at the permissive temperature, by a uvrD mutant, or by mitomycin C (MMC) or ultraviolet (UV) light treatment, was investigated. A deltapolB::kan mutant showed a significant decrease in the excision of Tn10 induced by the dnaB mutation, or by MMC or UV treatment, indicating the participation of Pol II in this type of deletion process. However, no effect of Pol II was evidenced in the excision of Tn10 stimulated by the uvrD mutation. The effect of the polB mutation on Tn10 precise excision induced by all these treatments was compared to that of mutations in repair-recombination genes recF and recA. The results reveal that the degree of participation of these genes varies depending on the agent that stimulates the deletion event.     

Postexcision transposition of the transposon Tn10 in Escherichia coli K12

An experimental analysis of the fate of transposon Tn10 after excision from a proA::Tn10 site localized on the plasmid F' leads to the conclusions: 1. The precise excision is a progressive process. Its probability is estimated per time unit. 2. An excised Tn10 is always integrated into a different genetic locus. 2. An excised Tn10 is always integrated into a different genetic locus. 3. The kinetics of postexcision transposition are sometimes very slow. The excised transposon is inherited in one cell line in spite of cell multiplication. 4. The processes of excision and secondary insertion have no absolute requirement for the recA+ genotype but they are strongly enhanced in recA+ cells. 5. The kinetics of postexcision transposition are strongly dependent on the genetic site from which the transposon was excised. 6. The probability of postexcision transposition is fully determined by the probability of excision and depends on the genotype of the host and many other factors.