INTRODUCTION

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A transposon is a DNA sequence that is able to insert itself into a new location in the genome without requiring any sequence similarity within the target DNA. Transposons cause genome rearrangements and are partially responsible for the spread of bacterial antibiotic resistance, heavy metal resistance, and increased tolerance to other deleterious agents (2). Furthermore, bacterial transposition reactions serve as paradigms for many gene transfer systems, including the processing and integration of the human immunodeficiency virus type 1 provirus (10, 17, 31). Therefore, understanding the mechanism of transposition and the role of host factors in this process is of considerable interest. Nonetheless, the involvement of host factors both in transposition and in retroviral integration is poorly understood.

Tn5 is a composite 5.8-kb transposon that contains 1.5-kb inverted repeats (IS50R and IS50L) (3, 38). IS50R and IS50L each have 19-bp repeats at their ends (the inside end and the outside end). These sequences are recognized by the transposase and by host factors involved in Tn5 transposition (3, 38). IS50R encodes two proteins that are important for transposition: a 476-amino-acid-long cis-acting transposase (Tnp) and a 421-amino-acid-long trans inhibitor (Inh) (3, 38). The coding sequences of Tnp and Inh are identical except that Inh lacks 55 N-terminal amino acids. IS50L encodes two proteins, P3 and P4, that have no known function. P3 and P4 have the same coding sequences as Tnp and Inh, respectively, except that both P3 and P4 lack 26 C-terminal amino acids. The central 2.75-kb region of Tn5 contains three cotranscribed antibiotic (kanamycin, bleomycin, and streptomycin) resistance genes. This region does not have a known function in transposition (3, 38).

Transposition is a very rare event in many organisms due to tight regulation whereby proteins (and/or functions) encoded by the transposon itself and, in some cases, specific host factors play a role (2, 38). For example, regulation of Tn5 transposition is dependent on the ratio between the two proteins, Tnp and Inh, encoded by the element itself (in IS50R). The Tn5 transposition frequency is also affected by a number of host factors (3, 38). Integration host factor (25), HU (3), DNA polymerase I (42), DnaA (55), topoisomerase I (Topo I) (46), and DNA gyrase (21) are probably involved in the positive regulation of Tn5 transposition, while Dam DNA methylase (56), Fis (51), and SulA (43) are negative regulators. However, the underlying mechanisms of the majority of these host factors in transposition remain to be determined.

Tn5 Tnp overproduction kills its host (52). It has been shown that Tnp overproduction causes filament formation and defective nucleoid segregation (52, 54). This phenomenon could be a consequence of Tnp interaction with a host factor(s) involved in transposition and a landmark event of the cell cycle, such as DNA segregation. Hence, Tnp overproduction killing could be used as a tool to study the host factors involved in Tn5 transposition and a possible relationship between transposition and chromosomal DNA segregation. Interestingly, Tnp overproduction killing does not require an active transposase (52). Nevertheless, analysis of N-terminal deletions of Tnp showed that deletion of the first 3 or 11 amino acids (in the 3 or 11 mutant) from the N terminus blocked killing while the same amount of Tnp accumulated in the cell (52). The wild-type N terminus of Tnp must, therefore, be critical for the cell killing phenotype.

To investigate the Tnp overproduction killing phenotype further, we isolated and localized four host mutations, designated stk, that suppress Tnp overproduction killing. These mutations were localized to four discrete loci in the Escherichia coli genome. The mutations map very close to genes known to be involved in cell division or DNA segregation: stkD10 at 28 min (shown in this report to be in topA, encoding Topo I), stkA14 at 76 min (in rpoH, encoding sigma 32 [54]), stkB33 at 85.5 min (near xerC, uvrD, and recQ), and stkC12 at 99.5 min (near dnaK, dnaJ, dnaT, and dnaC) (52).

Previously we have studied stkA14 and localized this mutation to rpoH. The sigma 32 mutation causes a constitutive induction of heat shock protein levels, suggesting that an induction of some sigma 32-programmed function(s) suppresses Tnp overproduction killing. However, none of the well-known heat shock functions appear to be involved in Tnp-associated killing (54).

In this report, we present evidence that Tnp killing may be due to titration of a specific host factor, DNA Topo I. The evidence comes from a detailed characterization of a second stk mutant, stkD10, localized in topA. This mutation causes a sixfold increase in Topo I abundance, and the mutant Topo I is only partially active. The mutation causes an alteration in gyrase activity. We also show that overproduction of wild-type Topo I partially suppresses Tnp overproduction killing. Additional evidence was obtained by a closer examination of stkA14. topA is transcribed at a higher rate in stkA14, increasing Topo I abundance and activity. These results suggest that the suppressor phenotype of this mutation is probably due to an increase in Topo I abundance. Finally, we present evidence (in partial agreement with Sternglanz et al. [46]) that DNA Topo I stimulates Tn5 transposition up to 30-fold in vivo. These results suggest that there is a relationship between Tnp overproduction killing and Topo I.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains and media. The E. coli K-12 strains used (Table 1) were grown in Luria broth (LB) (41) or M9-glucose minimal salt medium (41) containing all amino acids (except methionine and cysteine), each supplemented with the following five vitamins at 0.01 mM: p-aminobenzoic acid, p-dihydroxybenzoic acid, p-hydroxybenzoic acid, pantothenate (calcium salt), and thiamine (50). MgSO₄ was added to 0.1 mM (final concentration). LB plates contained 15 g of Bacto Agar per liter. The antibiotic concentrations were as follows: chloramphenicol, 20 µg/ml; ampicillin, 100 µg/ml; kanamycin, 40 µg/ml; streptomycin, 100 µg/ml; nalidixic acid, 5 µg/ml; and tetracycline, 15 µg/ml.

Plasmids. Most of the plasmids used in this study are detailed in Fig. 1. pRZ4775 (encoding Tnp under p_R control) was described by Weinreich et al. (52). pRZ4787 was described by Weinreich (53). pJW312-SalI, carrying topA (encoding Topo I), was obtained from J. Wang (57). Cloning vector pBII was supplied by A. Roca, and pBIP3, a cloning vector for allele exchange experiments, was obtained from R. Maurer (45).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Plasmids used in this study. pRZ4775, encoding wild-type Tnp, does not have IS50 end sequences; pRZ4787 encodes Tnp and contains a mini-Tn5 cassette; pRZ4775 does not express Tn5 Inh; pRZ4787 expresses Tn5 Tnp and Inh; pJW312-SalI encodes wild-type Topo I under Plac control. OE, outside end; IE, inside end; amp, ampicillin resistance; cam, chloramphenicol resistance; kan, kanamycin resistance.

topA allele replacement. Mutant and wild-type alleles of topA were subcloned into pBIP3 as described above. The plasmids were then transferred into E. coli, and a phagemid-based system for generating allele replacements was used as described by Slater and Maurer (45). After presumed allele exchange, about 200 to 250 individual colonies for each construct were tested for the transposase overproduction phenotype at 42°C. pRZ4775 was used as the Tnp overproducer in these experiments.

³⁵S labeling. The cells were grown in M9-glucose medium supplemented with all amino acids (except methionine and cysteine) and five vitamins (50) at 32°C to an optical density at 450 nm of 0.1. The cultures were then shifted to 42°C to induce Tnp overproduction and, after a 1-h induction, pulse-labeled by addition of 20 µCi of Trans³⁵S-label mixture (L-[³⁵S]methionine and L-[³⁵S]cysteine; 1.146 Ci/mmol; ICN Pharmaceuticals, Inc.) for 1 min with shaking; then the label was chased by addition of 100 µl of methionine and cysteine (each at 10 mg/ml) for 1 min. The labeled sample was mixed with 110 µl of 50% trichloroacetic acid (final trichloroacetic acid concentration of 5%). For each sample, (5 × 10⁵ cpm was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel.

Western blot analysis of Topo I. The overnight cultures of the wild type (MDW505) and an stkD10-containing strain (MDW565) were diluted 1:100 and grown to mid-log phase at 32°C (in the absence of Tnp) or at 42°C (in the presence of Tnp). The cells were harvested and sonicated (57). Equal amounts of proteins from all samples were separated by SDS-PAGE (10% gel) and transferred onto nitrocellulose filters (41). Western blots were performed as instructed by the manufacturer (DuPont NEN). The antibody for E. coli DNA Topo I was kindly supplied by J. Wang. The Western blots were scanned in a densitometer, and the intensities of the protein bands were quantitated by using ImageQuant and Excel programs.

Quantitation of Tnp overproduction killing by efficiency of plating experiments. After transformation of the plasmids (pRZ4775 and/or pJW312-SalI) into CaCl₂-treated wild-type (MC1061) competent cells (41), 12 to 20 individual colonies for each sample were grown overnight at 32 or 37°C in LB containing the appropriate antibiotic(s). Depending on the mode of regulating Tnp synthesis and/or various host proteins in individual experiments, dilutions of each sample were plated onto LB agar plates containing the appropriate antibiotic(s) and the indicated concentrations of isopropyl--D-thiogalactopyranoside (IPTG) (see figure legends) or were incubated at 32 and 42°C. After 18 h, the number of colonies formed in the presence of IPTG or at 42°C was compared with the number in the uninduced control samples (grown in the absence of IPTG or at 32°C).

PCR amplification and DNA sequencing of topA. The coding region topA and its control region were PCR amplified in four partially overlapping fragments from both stkD10 and wild-type (MC1061/pRZ4775) strains (see Fig. 4). The amplifications were carried out as described previously (54). The PCR-amplified fragments were sequenced with dye termination (PRISM; Applied Biosystems Inc.) by following the manufacturer's protocol. The sequencing reactions were analyzed at the University of Wisconsin Biotechnology Center. The presence of the mutation was confirmed by using four separate PCR stocks: two different stocks of fragment 1, reading the noncoding strand; and two different stocks of fragment 2, reading the coding strand.

Protein assays. Protein concentrations in cell extracts were determined by the Bradford protein assay as described by Rossomando (39).

Measurement of plasmid supercoiling. To determine plasmid DNA supercoiling in different stk mutants, the mutant and wild-type strains were transformed with pUC19. Plasmid DNA was isolated from overnight cultures by the alkaline method as described by Sambrook et al. (41). The plasmid DNAs were analyzed by 1% agarose gel electrophoresis in the presence of chloroquine (12 µg/ml). Electrophoresis was carried out as described by Mizushima et al. (30). The gels were stained with Syber Green II (Molecular Probes) and analyzed with a FluorImager (Molecular Dynamics).

Topo I relaxation assay. DNA Topo I relaxation activity was assayed by the ability of a crude lysate prepared from the stk and wild-type strains to relax supercoiled pUC19 DNA. The plasmid DNA was prepared by CsCl purification as described previously (41). The crude cell extract was prepared and the relaxation assay was performed exactly as described by Zumstein and Wang (57) except that the gels were stained with Syber Green II (Molecular Probes) and were examined with a FluorImager (Molecular Dynamics).

DNA gyrase activity assay. DNA gyrase activity was assayed by the ability of a crude lysate prepared from the stk and wild-type strains to introduce negative supercoils into relaxed pUC19 DNA in the presence of ATP. The same CsCl-purified pUC19 DNA as used in the Topo I relaxation assays was used, except that the DNA was relaxed by using wheat germ Topo I (Promega) as described by the manufacturer. The gyrase activity tests were carried out as described by DiNardo et al. (12), but with the same crude cell extracts as prepared for the Topo I relaxation assay. The gels were examined after staining with Syber Green II with a FluorImager.

Primer extension. The primer extensions were carried out as described by Sambrook et al. (41). The gels were analyzed with a PhosphorImager (Molecular Dynamics).

Transposition assays. The  infection assays were carried out as described by Sternglanz et al. (46) by using NK467. The stains were modified to obtain isogenic backgrounds as follows. An F' plasmid encoding lacI^q from E. coli XL-Blue was introduced into RS2, SD7, and JTT1. Then pJW312-SalI was introduced into the strains containing the F' plasmid. Six individual colonies for each strain (RS2F', RS2F'/pJW312-SalI, SD7F', SD7F'/pJW312-SalI, JTT1F', and JTT1F'/pJW312-SalI) were used in the  infection assay. RS2 and SD7 are topA10 strains with the same genotype. To ensure that the observed results were not due to genotypic changes during storage in a given strain, two different topA strains were used. For the same reason, two different topA strains (DM800 and a derivative) were also used. To determine the transposition frequency under Topo I overproduction conditions, strains containing pJW312-SalI (the plasmid encoding topA) were induced by using 0 to 0.2 mM IPTG. DM800 was transformed by pBAD33, so that the strain would contain cam as a selective marker. The F' plasmid from E. coli from XL-Blue containing lacI^q was introduced yielding HY126. This strain is topA. To obtain an isogenic topA⁺ strain, HY126 was then transformed with pJW312-SalI, resulting in HY152. For each strain, 16 individual colonies were used in the  infection assay.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Localization of stkD10 in topA. (i) stkD10 causes a five- to six-fold increase in the abundance of Topo I. Previously four stk (suppressor of transposase overproduction killing) mutations were isolated and located to four discrete loci in the E. coli genome (53). We studied these mutants further in order to investigate the host factors involved in Tn5 transposition and its regulation. This strategy might also help to determine whether there is a coupling between Tn5 transposition or its regulation and chromosomal DNA segregation, since Tnp overproduction causes defective DNA segregation (52, 54).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   stkD10 causes higher levels of three proteins of 90 to 100 kDa. Pulse-labeling and SDS-PAGE analyses were done as described in Materials and Methods; autoradiograms of the gels are presented. The autoradiograms were quantitated by densitometric analyses. All strains used, wild type (wt), MDW565 (stkD10), and MDW570 (stkA14), harbor pRZ4775 for Tnp overproduction. The molecular masses were estimated from the known molecular masses of Tnp (53 kDa), DnaK (70 kDa), and Hsp90. The gel shown is representative of three individual experiments.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   stkD10 mutation causes an increase in the level of Topo I. Western blot analysis of cell extracts (32 µg of total cell protein from each strain) prepared from wild-type (wt), stkD10-containing, and stkA14-containing strains was carried out as described in Materials and Methods with Topo I antibodies, and the results were analyzed by densitometry. (A) Cell extracts prepared from the wild-type strain and the stkD10-containing strain at 32°C (in the absence of Tnp); (B) cell extracts prepared from the wild-type strain and the stkD10-containing strain at 42°C (in the presence of Tnp); (C) cell extracts prepared from the stkA14-containing strain at 42°C (in the presence of Tnp) and the stkA14-containing strain at 32°C (in the absence of Tnp) and from the wild-type strain. Comparable Western blot experiments were performed four times with similar results.

(ii) stkD10 is located in topA (encoding Topo I). To determine the precise nature of the stkD10 mutation, we performed a sequence analysis of PCR-amplified topA and its control region (both from an stkD10-containing strain and from the parental wild-type strain) as shown in Fig. 4. Sequencing of these PCR-amplified fragments as described in Materials and Methods revealed that the stkD10 mutation lies in topA and results in an alanine-to-aspartate change at codon 118 (Fig. 4). We confirmed this mutation by sequencing four individual PCR-amplified stocks.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   stkD10 is located in topA. The topA region was PCR amplified and sequenced. The stkD10 mutation was localized in the overlapping region of fragments 1 and 2. The DNA and resulting amino acid sequence changes are shown. The presence of a mutation was confirmed by analyzing four different PCR stocks.

(iii) The topA mutation is sufficient for the suppressor phenotype caused by the stkD10 mutation. To investigate whether the topA mutation by itself is sufficient to suppress Tnp killing, we cloned wild-type topA onto a phagemid, pBIP3, and inserted the mutation stkD10 as described in Materials and Methods. The presence of the mutation was confirmed by sequencing the phagemid clone. We used the allele replacement method described by Slater and Maurer (45) to introduce the topA mutation and, as a control, the wild-type allele into a wild-type strain. After selecting appropriate recombinants, we tested the isolates for the Tnp overproduction killing. In four individual experiments in which 60 colonies were tested each time, we found that when the topA mutation was introduced into the wild-type strain, 38 (±2)% of the recombinants became mutant (98% resistant to Tnp overproduction killing) while 62 (±2)% remained wild type (99% sensitive to Tnp overproduction killing). When the wild-type clone was used, 98 (±2)% of the recombinants remained wild type (99% sensitive for Tnp killing). These results clearly show that the topA mutation is sufficient for the suppression of Tnp killing in the stkD10-containing strain.

Properties of the mutant Topo I. It is critical to examine the effects of the mutation (stkD10) on Topo I activities in order to understand the mechanism(s) for the suppression of Tnp killing. In addition, an understanding of the properties of the Topo I mutant may help elucidate a possible role for Topo I in Tn5 transposition.

(i) The mutant Topo I is defective for the DNA relaxation activity. Here, we present data showing that the stkD10 mutant version of Topo I is defective in its ability to relax negatively supercoiled DNA. This conclusion was made by examining DNA supercoiling of plasmid DNA isolated from the stkD10-containing strain as well as testing the crude cell extracts prepared from stkD10-containing and wild-type strains for Topo I relaxation activity.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   stkD10 causes a decrease in Topo I relaxation activity. Plasmid (pUC19) DNAs (2 µg) prepared from the wild-type (wt) and stkD10-containing strains were electrophoresed in the dark on a 1% agarose gel containing chloroquine (12 µg/ml). The gel was stained and analyzed with a FluorImager (Molecular Dynamics). Densitometric traces of the two lanes have been overlaid to show the differences in DNA supercoiling.

View larger version (45K): [in this window] [in a new window]   FIG. 6.   stkD10 causes reduced Topo I relaxation activity in vitro. Crude cell extracts (12 µg of crude cell protein/reaction) prepared from wild-type (wt), stkD10-containing and stkA14-containing strains at 32°C were used to relax supercoiled pUC19 DNA (0.5 µg/reaction) in vitro (57). The reactions were stopped (57) and run on a 1% agarose gel. The gel was stained and analyzed with a FluorImager (Molecular Dynamics). Densitometric traces of lanes 2 to 4 have been overlaid to show the differences in DNA supercoiling. Lane 1 contains the unreacted substrate DNA.

(ii) stkD10 confers a modest alteration in gyrase activity. The same crude cell extracts as used in the Topo I assays were used to determine DNA gyrase activity in both stkD10-containing and wild-type strains as described by DiNardo et al. (12) (Fig. 7). Quantitation of the gels indicated that DNA gyrase activity is reduced four- to fivefold in the stkD10 mutant. This was calculated by measuring the percentage of relaxed pUC19 DNA converted into supercoiled DNA. A densitometric scan of an overlaid graph of the gel is also shown in Fig. 7 to demonstrate the differences between the wild type and the stk mutants.

View larger version (48K): [in this window] [in a new window]   FIG. 7.   stkD10 and stkA14 mutations cause a modest alteration in gyrase activity. The same crude cell extracts (12 µg of crude cell protein/reaction) as used for Fig. 6 were used to measure gyrase activity in vitro. The percentage of pUC19 converted to supercoiled DNA was measured (12). One percent agarose gel electrophoresis was used, and the topoisomers were quantitated by densitometry. Lanes: 1, control DNA; lanes 2 and 5, DNA treated with a wild-type (wt) cell extract; 3 and 6, DNA treated with a cell extract from the stkD10-containing strain; 4 and 7, DNA treated with a cell extract from the stkA14-containing strain. The cell extracts used in lanes 5 to 7 were fourfold diluted. Densitometric traces of lanes 5 to 7 have been overlaid to show the differences in the DNA supercoiling.

Co-overproduction of wild-type Topo I partially suppresses Tnp killing. As mentioned above, there is in vivo evidence that Topo I is increased in stkD10 and stkA14. Therefore, we wanted to directly assess whether there is a relationship between suppression of Tnp killing and an increase in Topo I quantities. To test this possibility, we overproduced wild-type Topo I from P_lac by varying the concentration of IPTG (0 to 0.2 mM) to achieve different levels of expression while overproducing Tnp under the control of lambda p_R. The results in Fig. 8 (averages from separate experiments [the differences between experiments were less than 2%]) show that Topo I co-overproduction suppresses Tnp killing 100-fold. Although this level of suppression is substantially less than observed for the stkA14 or stkD10 mutant, its occurrence supports a link between Tn5 Tnp killing and Topo I level.

View larger version (15K): [in this window] [in a new window]   FIG. 8.   Co-overproduction of wild-type Topo I suppresses Tnp killing. Overnight cultures of MDW505/pJW312-SalI (+ Topo I) and MDW505 (wild type) were diluted and plated on LB agar containing various concentrations of IPTG (0 to 0.2 mM). After 18 to 14 h of incubation at 32°C (no Tnp induction) and at 42°C (Tnp induction), the colonies were counted and the percentage of CFU of the Tnp-induced cultures relative to the uninduced cultures was calculated. In general, the variation between different experiments for the same data points was less than 1%. IPTG induces overproduction of Topo I. For instance, Western blot analysis indicated a 10- to 12-fold increase in Topo I when cells were grown in the presence of 0.06 mM IPTG.

Possible connection between stkA14 and stkD10. (i) stkD10 and stkA14 both lead to an increase in total topA mRNA. Our previous studies regarding stkA14 (in rpoH) suggested that a sigma 32-regulated function other than induction of the well-known heat shock response proteins is responsible for the suppressor phenotype of stkA14. Since it has been shown that the P1 promoter of topA is sigma 32 dependent (23), we examined Topo I RNA levels in an stkA14-containing strain and an stkD10 containing strain. We used primer extension to determine the levels of the topA transcripts and to determine which promoters are responsible for the transcription in a given strain. Equal amounts of total RNA were subjected to primer extension (Fig. 9). The primer extension studies show that in the stkA14-containing strain, 70% of the mRNA is driven by the P1 promoter and the P1 RNA level is 10-fold higher than in the wild-type strain (Fig. 9, lanes 1 and 3). In addition there was two- to threefold increase from the P2 and P3 transcripts in the stkA14-containing strain. The total topA mRNA level in the stkD10-containing strain is about six- to sevenfold increased (lanes 1 and 2) and is driven mostly from P2 and P3. This could be explained by supercoiling-dependent regulation of topA transcription (47, 48). This finding of increased transcription of topA in the stkD10-containing strain clearly explains the increased abundance of the mutant Topo I. 

View larger version (39K): [in this window] [in a new window]   FIG. 9.   stkD10 (Topo I mutant) and stkA14 (sigma 32 mutant) alter topA mRNA levels. Primer extensions were done with equal amounts of total RNA (2 µg) isolated from wild-type (wt), stkD10-containing, and stkA14-containing strains. The P4 promoter of topA was used as an internal control. The gels were quantitated with a PhosphorImager (Molecular Dynamics). P1, P2, P3, and P4 indicate the positions of known topA transcripts. The sequencing standard (G, A, T, and C) was a dideoxy primer extension analysis of wild-type topA.

(ii) stkA14 causes an increase in both DNA gyrase and Topo I activities. Crude cell extracts prepared from the stkA14-containing strain were used to determine Topo I relaxation activity in vitro as described by Zumstein and Wang (57) (Fig. 6, lane 3). Figure 6 also shows a densitometric scan of lane 3. The results (averages from three separate experiments) showed that there is about a four- to fivefold increase in the topoisomerase relaxation activity in the stkA14-containing strain. This result correlates with the Topo I protein level in that strain.

DNA topology is not critical for suppression. If an alteration of DNA topology is required to suppress Tnp overproduction killing, one would expect to see a similar pattern of in vivo DNA topology in all suppressor strains. We compared the in vivo plasmid DNA supercoiling in all stk mutants that were isolated (52, 53) and with that of the same plasmid DNAs isolated from the wild type.

View larger version (107K): [in this window] [in a new window]   FIG. 10.   DNA topology is not critical for suppression. Plasmid (pUC19) DNAs (0.5 to 2 µg) prepared from wild-type (wt) and stk mutants were electrophoresed in the dark on a 1% agarose gel containing chloroquine (12 µg/ml). The gel was stained and analyzed with a FluorImager (Molecular Dynamics).

Topo I is a stimulator of Tn5 transposition. If Topo I plays an essential role in Tn5 transposition, then topA-defective mutations should dramatically affect the frequency of Tn5 transposition. Sternglanz et al. (46) have reported that Tn5 transposition decreases 70- to 90-fold in topA strains and about 40-fold in topA10 strains. These strains were subsequently shown to have compensatory mutations in DNA gyrase. Therefore, we have reexamined the effects of both topA and topA10 on Tn5 transposition, using control strains that are as nearly isogenic with the topA mutants as possible.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Tnp killing is due to a titration of Topo I. Detailed examination of stkD10 and stkA14 mutants strongly suggests that their suppressor phenotypes are due to an increase in Topo I levels. stkD10 is mutated for topA. The mutant Topo I has reduced relaxation activity (Fig. 5 and 6). This reduction is likely responsible for the increase in topA transcription, which then causes the increased level of Topo I. This is due to the supercoiling-dependent regulation of topA transcription (47, 48). The reduced DNA gyrase activity (Fig. 7) in this mutant can be explained by the supercoiling-dependent regulation of gyrase transcription (16, 26).

Topo I modulates Tn5 transposition. The examination of stkD10 and stkA14 mutants suggested that Topo I is involved in Tnp killing. Sternglanz et al. (46) presented evidence that Topo I enhances Tn5 transposition; however, it was later suggested that these results were due to the compensatory DNA gyrase mutants in these backgrounds (3).

Could Topo I titration by Tnp cause a defect in DNA segregation? The results presented here suggest that Tnp overproduction killing could be due to a specific interaction between Tnp and Topo I. We have also shown that Tnp overproduction causes defective DNA segregation and an increase in anucleated cell formation (52, 54). It has been shown that anucleated cells form in par (gyrB [40] and parC [1, 40]), mukA (19, 20), mukB (15, 34), minB (6), and xerC (4, 5) mutants. These gene products have been suggested to be involved in the bacterial chromosome segregation processes. Recently it has been shown that topA mutants can suppress mukB mutants (44) and overproduction of topA can suppress parC mutants (22, 35). These results suggest that Topo I could be involved in chromosomal DNA segregation, even though studies of the four topoisomerases of E. coli have suggested that Topo I is incapable of catalyzing decatenation in vitro (18, 49).