Characterization of the uup locus and its role in transposon excisions and tandem repeat deletions in Escherichia coli

Null mutations in the Escherichia coli uup locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn10 and to reduce the plaque size of bacteriophage Mu (Uup(-) phenotype). By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup(-) phenotype is the consequence of the absence of expression of the downstream gene (uup) of a two-gene operon, caused either directly by insertions in uup or indirectly by the polar effect of insertions in the upstream gene (ycbY). The promoter for uup was mapped upstream of ycbY by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter. The uup mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn10 derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision. Finally, we found that mutations that increase the frequency of precise excision of Tn10 are divisible into two categories, depending upon whether they did (uup, ssb, polA, and topA) or did not (mutHLS, dam, and uvrD) also increase precise excision frequency of the mini-Tn10 derivatives. It is suggested that the differential response of mini-Tn10 and Tn10 to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them.     

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.     

Evidence for Campbell's mechanism for fine excision of Tn9 type transposons in Escherichia coli K12]

The plasmid-transposon Tn9-322 was constructed by inverted transposition from the pBR322::Tn9 plasmid. The precise excision of the Tn9-322 transposon from the proB gene site can proceed by the Campbell's model. This fact was demonstrated by appearance of the plasmid-transposons after their precise excision. They contain two IS1 elements flanking a short direct repeat of the target DNA. The recombinational mechanism of precise excision of Tn9 type transposons seems not to be alternative but looks as an additional one to a well-known slippage mechanism proved for Tn5 and Tn10.     

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.     

The effect of mutations in recB and recC genes on the precise excision of Tn5 from pNM1 plasmid genome

The affect of mutations in chromosomal genes determining the realization of RecBC and RecF pathways of recombination in E. coli K12 on the frequency of transposon Tn5 precise excision from the genome of the conjugative plasmid pNM1 has been demonstrated. The pNM1 plasmid is a derivative of R100.1 and differs from the latter in the presence of Tn5 inactivating the tet gene of transposon Tn10.     

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.     

Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.     

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.     

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.     

Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis.

Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB.     

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.     

Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens.

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.     

Genetic analysis of Staphylococcus aureus with Tn4001.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.     

Hfr formation directed by tn10

The transposable drug-resistance element, Tn10, can serve as a region of homology to direct the insertion of an F'ts114 lac plasmid into the chromosome of Salmonella typhimurium. Derivatives of F'ts114 lac were constructed that carry Tn10 insertions; these plasmids were transferred to strains having a Tn10 insertion in the chromosome. Under these circumstances, Hfr formation requires homologous recombination between plasmid-borne and chromosomal Tn10 elements. The process is dependent on recA function and on the presence of both Tn10 elements. All Hfr's isolated from a given merodiploid show the same direction of transfer. Depending on the orientation of Tn10 in the F' plasmid, Hfr's transferring in either direction can be obtained from any chromosomal Tn10 insertion. Since Tn10 insertions can be generated in any region of the chromosome, this method permits the isolation of Hfr's with either direction of transfer having their origin at almost any predetermined site. The Hfr's constructed by this method are sufficiently stable for standard genetic mapping crosses, and they have also been used to generate new F' plasmids. Implicit in the results above is the possibility of determining the orientation of any chromosomal Tn10 insertion by constructing an Hfr using a standard F' Tn10 plasmid and determining the direction of chromosome transfer. The general approaches described here are applicable to other transposable elements and other bacterial systems.     

RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli

Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.     

Mismatch Repair Mutations of ESCHERICHIA COLI K12 Enhance Transposon Excision

Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.     

Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the ipaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 NotI restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence-associated chromosomal loci.     

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.     

Use of transposon-transposase complexes to create stable insertion mutant strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10(-8) +/- 0.87 x 10(-8) per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

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.