INTRODUCTION

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One of the features of mutations generated by the insertion of transposable elements is their ability to undergo true reversion, at characteristically low frequencies, by precise excision between the pair of host sequence-derived direct repeats that flank each insertion. Unlike other properties associated with transposons, precise excision is mediated by host-encoded functions and does not depend on the transposase encoded within each element (10, 13). In studies with the tetracycline resistance element Tn10 (13, 25, 26), Kleckner and coworkers have identified precise excision as one of three related genetic rearrangements, the other two being nearly precise excision (in which a deletion event between two repeats internal to Tn10 results in excision of all but 50 bp of the element, so that the target gene remains nonfunctional but there is relief of polarity on the expression of downstream genes in the operon) and precise excision of the 50-bp remnant of nearly precise excision. All three rearrangements are RecA independent and fall into the category of illegitimate recombination events.

The mechanism by which precise excisions occur is not known, nor is it clear what, if any, are the other non-transposon-related mutations, resulting from illegitimate recombination events in bacteria, that are mechanistically related to precise excision. Foster et al. (13) had provided early evidence that, whereas precise excision and nearly precise excision of Tn10 may occur by very closely related pathways, precise excision of the 50-bp remnant appears to occur by a different mechanism. One model has been that precise excision occurs by a RecA-independent replication slippage event across the pair of direct repeats (of host-derived sequence) flanking the insertion and that the inverted repeats at the ends of the element facilitate the process (10, 13, 48). The inverted repeats may, for example, participate in formation of intrastrand stem-loop structure(s), although alternative structures involving interactions between the inverted repeats as duplex DNA have not been excluded (10, 13, 40, 48). The former possibility is supported by the findings that the frequencies of precise and nearly precise excision are increased under conditions where the single-stranded template (which would more readily be able to form the stem-loop structure) is expected to be abundant, such as in the presence of an M13 ori sequence on the template (7) or during Tra-dependent synthesis of single-stranded DNA during conjugal transfer of an F' plasmid (30, 49). An in vitro model that mimics precise excision and that is mediated by replication slippage has also been reported (6). Finally, a separate phenomenon of UV-induced transposon precise excision that appears to require functions encoded by the SOS regulon has also been described (22, 34).

Mutations (designated tex, for transposon excisions) in several host genes that increase the frequency of precise excisions have been identified (16, 25, 26, 38). One such locus is uup (16, 38), which maps at 21 min on the Escherichia coli chromosome and increases the precise excision frequency of both transposons Tn5 and Tn10. Mutants in uup also exhibit a reduction in lytic growth of Mu bacteriophage. In an earlier study (38), we had described the isolation of several independent insertion mutations in the uup locus. Molecular cloning, complementation analysis, and nucleotide sequence determination of the gene identified by one of the disruptions had indicated that the Uup protein is cytosolic and belongs to the superfamily of ATP-binding cassette domain proteins (23).

In the present study, we have investigated the organization and regulation of the uup locus. Our results indicate that uup is part of a complex operon and that it is situated downstream of a conserved gene of apparently unrelated function (ycbY) with which it is cotranscribed from a moderately active, constitutive promoter. We also report that uup mutations increase the frequency of two other RecA-independent recombination events, namely, nearly precise excisions and deletions of one copy of a chromosomal tandem repeat (tandem repeat deletion). Finally, our results permit classification of the tex mutations into two categories, depending upon their differential effects on Tn10 derivatives with imperfect versus perfect terminal inverted repeats.

	MATERIALS AND METHODS

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Growth media and conditions. The defined and nutrient media were, respectively, minimal A medium (supplemented with glucose or other indicated C source and the appropriate auxotrophic requirements) and Luria-Bertani medium (31). Concentrations of antibiotics and 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) used were as earlier described (37, 38).

Bacterial strains and plasmids. The genotypes of E. coli strains used in this study are listed in Table 1. Plasmids were constructed from the following vectors: the high-copy-number derivatives pBluescript II KS (pBKS; Stratagene, La Jolla, Calif.) and pET21b (Novagen, Madison, Wis.); a medium-copy-number pSC101 derivative, pCL1920 (21); and a very-low-copy-number IncW derivative, pMU2385 (51), carrying the lacZ reporter gene for promoter cloning experiments. The extent of uup locus carried on each of the plasmids is depicted in Fig. 1.

DNA methods. The standard protocols of Sambrook et al. (42) were followed for experiments involving recombinant DNA, including plasmid manipulations, gel electrophoresis, transformation, preparation of radiolabeled probes, Southern blot hybridization, and DNA sequence determination on double-stranded plasmid DNA templates. The oligonucleotide primer 5'-TGGTCACCAACGCTTTTCCCGAG-3', designed so as to read outward from a site immediately internal to the right terminal inverted repeat of Tn10dTet2 (see Fig. 1), was used to determine the junction sequences of insertions generated with this element.

Construction of a chromosomal ycbY deletion-insertion mutant. A 1.2-kb BclI fragment that spans the promoter and proximal third of the ycbY open reading frame (ORF) was excised from plasmid pHYD633, and in its stead was ligated a 2.7-kb BamHI fragment (derived from Tn10dTet2) comprising the tetA and tetR genes. The resulting plasmid pHYD650 thus carries both a deletion and a tetracycline resistance insertion in ycbY. The ycbY::Tet mutation was recombined into the chromosome of the recD strain KM22 as described elsewhere (33), following transformation with 1 µg of a gel-purified 5.3-kb fragment from pHYD650 that carries the mutation and flanking DNA from the ycbY-uup locus. In order to control for the possibility that the mutation might be lethal, the linear transformation with the fragment from pHYD650 was attempted in both KM22 and KM22/pHYD646 (where pHYD646 is expected to provide ycbY⁺ and uup⁺ functions even after disruption of the chromosomal locus); equal numbers of Tet^r transformants were obtained with both recipient strains. A P1 lysate prepared on one KM22 Tet^r transformant, GJ2240, was then used to transduce the lacZ::Tn10dKan strain GJ1885, as well as the pHYD646 derivative of GJ1885, to Tet^r. Once again, Tet^r transductants were obtained in both strains at equal frequencies, and a 100% linkage was observed in GJ1885 between Tet^r and the Uup phenotype (data not shown). One such GJ1885 derivative was designated GJ2255.

Measuring mutation frequencies. In strains where reversions to lacZ⁺ were being examined, Lac⁺ papillation tests (37, 38) were employed to obtain rapid and qualitative estimates of mutation frequency. The general procedure for quantitative determination of mutant frequencies in cultures was as described by Fijalkowska and Schaaper (12). Briefly, the mutant frequency was calculated as the ratio of the median number of mutants in a series of (four to eight) cultures to the average number of viable cells per culture. The median frequency was chosen so as to avoid the problem of disproportionate contributions by mutational jackpots in individual cultures. In agreement with earlier reports (12, 13), threefold or greater differences in mutation frequencies between different strains were clearly reproducible in these experiments. In all the experiments involving selection for utilization of lactose, melibiose, or phenyl--D-galactoside as C source, appropriate minimal plates prespread with 10⁹ cells of the lac strain MC4100 (or its derivatives carrying the plasmid vector pBKS, pCL1920, or pMU2385 for surviving appropriate antibiotic supplementation) as scavenger were used.

att-gal

Isolation of a topA insertion mutant as tex. Random insertions of transposon Tn10dTet2 were generated in the chromosome of the lacZ::Tn10dKan strain GJ1885 after infection with the transposon vehicle phage NK1323, as described elsewhere (17). Tet^r colonies were screened on Luria-Bertani-lactose-X-Gal agar plates by the method earlier described (38), for clones that exhibited increased Lac⁺ papillation frequency following precise excision of the Tn10dKan insertion in lacZ. One tex mutant so identified also showed extremely poor growth characteristics on both defined and rich media and was designated GJ2243. The Tn10dTet2 insertion was cloned as part of a 12-kb PstI fragment from the chromosome of GJ2243 in the plasmid vector pCL1920, and the resulting plasmid was designated pHYD661. A radiolabeled probe prepared from DNA of plasmid pHYD661 hybridized to the recombinant  phages 253 and 254 of the ordered Kohara miniset phage library (20, 41). Data from physical mapping of pHYD661 permitted the inference that the Tn10dTet2 insertion in GJ2243 is located in topA (encoding topoisomerase I). Determination of the junction sequence (using pHYD661 as template) confirmed that the insertion had occurred immediately preceding the first base of codon 481 in the 865-residue-long topA ORF. The insertion allele in GJ2243 has been designated topA72::Tn10dTet2.

Other techniques. Procedures for transduction with P1 phage (15); determination of burst size following phage Mu c(Ts) infection (16); IPTG-mediated overexpression, and identification by gel electrophoresis, of the products of genes cloned into pET plasmid vectors and introduced into the BL21(DE3) strain (47); and measurement of -galactosidase activities in cultures (31) were as described previously. Strains were made recA following transduction either to Cm^r with a P1 lysate prepared on strain GJ1934 that carries a Tn10dCm insertion 60% linked to the recA locus or to Kan^r with a lysate prepared on a recA::Kan mutant.

	RESULTS

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Insertions in both uup and ycbY confer a Uup phenotype. In an earlier study (38), we had described the isolation and mapping by phage P1 transduction of three independent Tn10dTet insertion mutations (uup-351, 352, and 353) to a single chromosomal locus. The cloning, physical mapping, and sequence analysis of the gene designated uup that had been rendered null by the uup-351::Tn10dTet1 insertion was also described. Plasmids bearing the cloned uup⁺ gene complemented all three mutants, and conversely, a plasmid with the uup-351 insertion complemented none of them.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Extents of insert DNA from E. coli uup locus in plasmids used in this study. On top is depicted, to the indicated scale, the position of recognition sites for the enzymes BamHI (B), BclI (Bc), ClaI (C), EcoRI (E), HindIII (H), PstI (P), and SalI (S); for BclI, ClaI, and SalI, only the relevant sites have been marked. Also depicted are the positions of the uup-351, -352, and -353::Tn10dTet insertions (as inverted triangles). Immediately beneath is depicted the alignment of the ycbY, uup, pqiA, and pqiB' ORFs and the promoters (as hooked arrows) within uup and upstream of ycbY identified, respectively, by Roe's group (18) and in this study. The uup locus sequence is from the work of Blattner et al. (4) and is corrected from that reported earlier (18, 38). The inset shows a map, drawn to one-half scale, of Tn10dTet in the orientation present in each of the three uup insertions, and the solid bar marks the fragment used for radiolabeled probe preparation in the Southern blot of Fig. 2; the inset map is that of Tn10dTet2 present in uup-352 and uup-353, whereas the Tn10dTet1 element in uup-351 lacks the pair of BamHI sites shown (17, 38). Each line aligned beneath the uup locus physical map represents the extent of chromosomal DNA, delimited by the cut sites marked, that has been cloned into a plasmid(s) whose numerical pHYD designation(s) (and vector derivation[s] in parentheses) is indicated alongside. Abbreviations for plasmid vectors: pCL, pCL1920; pET, pET21b; and pMU, pMU2385. The interrupted line segment in the insert of pHYD650 depicts deletion of DNA between the parenthetical BclI sites marked, and the filled rectangle indicates insertion at this site of the 2.7-kb Tetr-encoding BamHI fragment from Tn10dTet2 (see inset). Digestions at the BclI sites and at the right ClaI site marked were done on DNA prepared from a dam strain.

View larger version (92K): [in this window] [in a new window]   FIG. 2.   Mapping of uup-352 and -353 insertions. Reproduced is the autoradiograph following Southern blot hybridization to electrophoresed DNA from strains GJ1887 (uup-352) and GJ1888 (uup-353) after digestion with EcoRI (E), EcoRI-PstI (EP), or HindIII-PstI (HP) of a radiolabeled probe prepared from the EcoRI-HindIII Tn10dTet fragment described for Fig. 1. At the left are shown the positions of migration of DNA markers of the indicated size in kilobases. Calculated sizes (in kilobases) of the hybridizing fragment for each digest of the uup-352 and uup-353 mutants were, respectively, 3.2 (E), 1.5 (EP), and 10.1 (HP) and 6.0 (E), 4.2 (EP), and 7.2 (HP).

View larger version (99K): [in this window] [in a new window]   FIG. 3.   Polypeptides encoded by the ycbY-uup operon. Protein extracts prepared from uninduced () and IPTG-induced (+) cultures of BL21(DE3) derivatives carrying the plasmid pET21b, pHYD653, or pHYD654 were subjected to gel electrophoresis on lanes as indicated and visualized by staining with Coomassie blue. Lane 1 represents marker proteins of indicated sizes in kilodaltons. Solid and open arrows identify bands of 73,000 and 47,000, respectively, in Mr that are discussed in the text.

ycbY expression is not required for Uup⁺ phenotype. In the next set of experiments, we examined whether the Uup phenotype associated with the ycbY insertion was because of (i) merely a polarity effect of the insertion on expression of the downstream uup gene or (ii) the need for ycbY as well in conferring the Uup⁺ phenotype. For this purpose, we constructed a pair of plasmids (pHYD642 and pHYD643) in which a fragment carrying uup⁺ without an intact ycbY had been cloned in either orientation into a site in the vector pCL1920. Both plasmids were able to complement the transposon precise excision phenotype of the mutant carrying the uup-353 insertion in ycbY (Table 2) as well as its Mu plaque size phenotype, suggesting that ycbY itself is not required for the Uup⁺ function.

Characterization of the ycbYuup operon promoter. We cloned several fragments from the ycbY-uup region upstream of the lacZ reporter gene in a single-copy-number plasmid, in order to examine promoter activities and regulation in vivo. Each of the fragments extended either upstream or downstream of the EcoRI site that cleaves at codon 2 of ycbY. Two upstream fragments (in plasmids pHYD632 and pHYD647) that extended respectively up to a ClaI site (0.29 kb) and an EcoRI site (1.2 kb) exhibited promoter activities of comparable strength (Table 3), suggesting that the promoter for the ycbY-uup operon is situated downstream of the ClaI site. Experiments of primer-extension analysis on total cellular RNA from uup⁺ strains (data not shown) were also consistent with the existence of a single transcription start site 172 bases upstream of the ycbY ORF.

Effect of uup on other types of genetic rearrangements. We had earlier shown that the frequency of occurrence of spontaneous point mutations to rifampin resistance or nalidixic acid resistance is not altered in uup mutant strains (38). We have now examined the effect of uup on several other types of spontaneous genetic rearrangements that fall under the broad category of RecA-independent recombination events, using the assays described above. We observed that the frequencies of nearly precise excision of Tn10dKan, as well as of tandem repeat deletion in lacZ, were elevated approximately four- to ninefold in both the recA⁺ uup and recA uup strains (Table 4). On the other hand, the uup mutations did not affect the frequencies of occurrence of (i) spontaneous mutations in lacI (roughly 70% of which are caused by insertion or deletion of a 4-bp sequence at a site where this sequence is present in three tandem repeats in the lacI⁺ gene [11, 45]), (ii) deletions in the att-gal locus, or (iii) precise excision of the 50-bp remnant following nearly precise excision of lacZ::Tn10dKan (data not shown). The last finding is consistent with an earlier report that precise excision of the Tn10 remnant might occur by a mechanism which is different from that mediating precise excision or nearly precise excision (13).

uup⁺ gene dosage effect on genetic rearrangements. While undertaking the complementation experiments, we observed that strains carrying the multicopy uup⁺ plasmid pHYD627 exhibited lower frequencies of precise excision (approximately 20- to 30-fold) than did the isogenic strain GJ2258 that was haploid uup⁺ (Table 2). The frequencies of nearly precise excision and of tandem repeat deletion were also reduced around 30- to 50-fold in the multicopy uup⁺ strains bearing plasmid pHYD627, compared to the values in strains carrying the vector pBKS (Table 4). Furthermore, the plaque size of Mu c(Ts) when plated on the multicopy uup⁺-bearing strain was significantly larger than that on the haploid uup⁺ strains (data not shown). The relevance of these observations to our understanding of the possible physiological role and mechanism of uup function is discussed below.

tex mutations fall into two categories. In the course of these studies, we have also found that the various tex mutations described so far can be classified into two categories (Table 5). An example of the first category is uup, which increases the precise excision frequency of both Tn10 (16) (Table 5) and the mini-Tn10 derivatives such as Tn10dKan (38) (Table 2), Tn10dTet (Table 5), and Tn10dCm (data not shown). Other examples of the first category include mutations in ssb (38) (Table 5) and polA (27) (Table 5), the genes encoding the single-stranded DNA binding protein SSB and DNA polymerase I, respectively.

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	DISCUSSION

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Operonic arrangement at the E. coli uup locus. The following lines of evidence obtained in this study establish that uup is cotranscribed with another ORF (ycbY) from a promoter situated upstream of ycbY. (i) An insertion mutation in chromosomal ycbY confers a Uup phenotype, because of a polarity effect on uup expression. (ii) Likewise, a plasmid (pHYD638) carrying the ycbY-uup locus with the insertion in ycbY failed to complement chromosomal uup mutations. (iii) Only one promoter capable of transcribing uup⁺ in the haploid state was identified in experiments involving either genetic complementation or lacZ reporter gene expression, and this promoter is situated upstream of ycbY. (iv) Finally, the coordinate production of proteins corresponding to both the ycbY and uup ORFs in the T7 RNA polymerase-based expression system is consistent with the notion that they constitute a single transcriptional unit. The ycbY-uup genes are also part of a more complex operon that includes downstream genes such as pqiA and pqiB for which additional promoters exist that are embedded in the uup ORF (Fig. 1).

A shared step in pathways of transposon excisions and tandem repeat deletions? We found that uup mutations increase the frequency of three different RecA-independent genetic rearrangements, namely, transposon precise excisions and nearly precise excisions and deletions of a tandem chromosomal repeat. This observation implies the existence of a shared step in the pathways of these events which is influenced by the Uup product. That precise excision and nearly precise excision share similar mechanisms has previously also been suggested by Kleckner and coworkers on the basis of other lines of genetic evidence (13, 25, 26).

Two categories of tex mutations. It has been shown earlier (10, 13) that the frequency of precise excision is determined in part by the length and degree of matching of the inverted repeat sequence at the ends of the transposable element. Other workers have also shown that the occurrence of spontaneous deletions in vivo and in vitro between short stretches of direct repeats is facilitated by the presence of palindromic sequences within the deletion interval (1, 6, 8, 35, 50, 53). These results have led to the hypothesis that an interaction between the inverted repeats represents an intermediate in the precise excision (or deletion) pathway.