Notice: Wiley Online Library will be unavailable on Saturday 27th February from 09:00-14:00 GMT / 04:00-09:00 EST / 17:00-22:00 SGT for essential maintenance. Apologies for the inconvenience.
The cellular function of Escherichia coli topoisomerase III remains elusive. We show that rescue of temperature-sensitive mutants in parE and parC (encoding the subunits of the chromosomal decatenase topoisomerase IV) at restrictive temperatures by high-copy suppressors is strictly dependent on topB (encoding topoisomerase III). Double mutants of parEΔtopB and parCΔtopB were barely viable, grew slowly, and were defective in chromosome segregation at permissive temperatures. The topB mutant phenotype did not result from accumulation of toxic recombination intermediates, because it was not relieved by mutations in either recQ or recA. In addition, in an otherwise wild-type genetic background, ΔtopB cells treated with the type II topoisomerase inhibitor novobiocin displayed aberrant chromosome segregation. This novobiocin sensitivity was attributable to an increased demand for topoisomerase IV and is unlikely to define a new role for topoisomerase III; therefore, these results suggest that topoisomerase III participates in orderly and efficient chromosome segregation in E. coli.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Topoisomerases are essential enzymes that regulate and maintain the topological state of chromosomal DNA by making a transient DNA break, passing a DNA segment through that break, and subsequently resealing the break (Wigley, 1995; Champoux, 2001; Bates and Maxwell, 2005). Type I topoisomerases cleave and pass one strand of DNA, whereas type II enzymes cleave and pass both strands of DNA (Wigley, 1995; Champoux, 2001; Bates and Maxwell, 2005).
Topoisomerases are essential for managing DNA topology during all macromolecular transactions on chromosomal DNA. In particular, their activities ensure the removal of excess positive topological linkages that accumulate during DNA replication because of the unwinding of the parental DNA strands. Either a type I or type II enzyme can be effective at this task. Once replication is completed, any remaining linkages between the two parental strands are manifested as catenanes between the two daughter chromosomes. If the replicated strands are completely sealed, then only a type II enzyme can unlink the daughter molecules (Sundin and Varshavsky, 1980; 1981; Wang, 1991). Champoux and Been (Champoux & Been, 1980) noted that the excess positive linkages that accumulate ahead of the replication fork can diffuse behind the replisome to form windings of the partially replicated daughter duplexes about each other that have come to be called precatenanes (because once replication is completed they convert to catenanes). We demonstrated that precatenanes do form during replication of a circular DNA in vitro (Peter et al., 1998). Precatenanes can clearly be resolved by type II topoisomerases, however, we have suggested that a type I topoisomerase operating at or near the replication fork where single-stranded gaps are available could also unlink them (Nurse et al., 2003).
Escherichia coli harbours four DNA topoisomerases: two type I enzymes [topoisomerase (Topo) I and III] and two type II enzymes (DNA gyrase and Topo IV) (Bates and Maxwell, 2005). Extensive studies and the properties of these enzymes have implicated some of them in clearly defined roles in the cell. DNA gyrase (encoded by gyrA and gyrB) (Gellert et al., 1976a; Mizuuchi et al., 1978) is required for preserving the global negative supercoiled state of the chromosome (Champoux, 2001), which it does primarily via the conversion of positive supercoils generated by DNA replication and transcription directly to negative supercoils (Levine et al., 1998; Wang, 2002). Topo I (encoded by topA) is important for preventing the accumulation of excess negative supercoils by specifically relaxing negatively supercoiled DNA (Wang, 1991), thus joining with DNA gyrase to homeostatically regulate the global level of chromosomal supercoiling (Menzel and Gellert, 1983).
Topo IV is required for the complete topological separation of the linked daughter chromosomes generated by DNA replication and is essential for viability (Kato et al., 1990; Adams et al., 1992; Peng and Marians, 1993). Topo IV consists of a heterotetramer of ParE and ParC dimers (Kato et al., 1992; Peng and Marians, 1993). Temperature-sensitive mutants of parE and parC manifest a partitioning defect (par) (Kato et al., 1989; 1990) where cell division is arrested, leading to extreme filamentation coincident with the accumulation of one or two large masses of unsegregated, replicating DNA at the cell centre.
More is known about the activities of Topo III in vitro than its role in vivo. This enzyme binds tightly to single-stranded DNA and does not appear to have the ability to locally unwind duplex DNA to create a binding site for catalysis. Thus, it is a potent decatenase on DNA rings that have small gaps, but is very poor at relaxing supercoils (Srivenugopal et al., 1984; DiGate and Marians, 1988), suggesting that it is unlikely to contribute to the maintenance of global supercoiling. On the other hand, Topo III can support replication fork progression in the absence of either DNA gyrase or Topo IV, and generate completely unlinked daughter chromosomes (Hiasa and Marians, 1994). The ability of the enzyme to remove precatenanes is thought to contribute to these activities (Nurse et al., 2003).
Mutants of topB (encoding Topo III) are viable and do not display obvious chromosome abnormalities or growth defects, thus it is not clear if Topo III is capable of resolving catenanes in vivo, but these mutants do show an increase in deletions between short homologous regions and an increase in frameshift mutations, suggesting that cells lacking topB have either an increased incidence of recombination at short DNA repeats or DNA replication mispairing slippage (Yi et al., 1988; Schofield et al., 1992; Uematsu et al., 1997). In addition, topB mutations are synthetically lethal with topA (Zhu et al., 2001) and parE mutations (Lopez et al., 2005) and it has been reported that deletion of recA rescues the synthetic lethality in both cases, suggesting a role for topB in the resolution of recombination intermediates (Zhu et al., 2001; Lopez et al., 2005). This observation is consistent with the known role of Topo III in eukaryotes, where it has been shown to associate with various RecQ family helicases in resolving toxic, unscheduled recombination intermediates that can occur, for example, at stalled replication forks (Oakley and Hickson, 2002). Failure to resolve such intermediates can lead to chromosome breakage, chromosome rearrangements, and faulty chromosome segregation.
Functional cooperation between the E. coli enzymes has been demonstrated in vitro as well: Topo III and RecQ can catenate covalently closed, double-stranded DNA rings (Harmon et al., 1999) and catalyse the resolution of stalled, converging replication forks (Suski and Marians, 2008). Collectively, these data point to a role for Topo III in vivo of either clearing toxic recombination and replication intermediates or unlinking sister chromosomes during replication, possibly with the assistance of its cognate helicase RecQ.
To understand how Topo IV could be regulated and to identify other proteins involved in resolving topologically linked sister chromosomes, we identified high-copy suppressors of the temperature-sensitive (ts) alleles parE10 and parC1215. Overproduction of several replisome components: the γ and τ subunits of the DNA polymerase III holoenzyme (both encoded by dnaX) and the β processivity clamp (encoded by dnaN), as well as an integral membrane protein encoded by setB, and Topo III could suppress the Topo IV mutant par and growth defects at non-permissive temperatures (Levine and Marians, 1998; Nurse et al., 2003; Espeli et al., 2003b).
As a potent decatenase (DiGate and Marians, 1988), it was reasonable to speculate that Topo III, when overproduced, could suppress parE and parC ts mutations at the restrictive temperature by decatenation of linked sister chromosomes. However, the question remained: Does Topo III participate in the removal of catenanes and/or precatenanes under physiological conditions?
Suppressors of the parE10 and parC1215 mutations are likely to either play a role in chromosome segregation or influence Topo IV activity. Here we have investigated the role of these suppressors. We find that topB is required for the suppressors to promote growth of parE and parC ts mutant strains at restrictive temperatures, suggesting that the mechanism of suppression could be potentiation of Topo III activity. Combinations of ΔtopB with the Topo IV ts mutations were synthetically lethal at permissive temperatures and these double mutant strains exhibited chromosome segregation defects. Contrary to previously reported findings (Lopez et al., 2005), neither recQ nor recA were required for the parE10ΔtopB synthetic lethality, because mutations in recQ and recA did not rescue the parE10ΔtopB double mutant strain. Furthermore, in the absence of these recombination proteins, the high-copy suppressor γ [dnaX(γ)] is still unable to rescue the parE10ΔtopB double mutant strain at 30°C, arguing that the observed phenotype was not the result of the accumulation of toxic recombination intermediates. However, we discovered that mutants of topB are hypersensitive to the type II topoisomerase inhibitor novobiocin: when exposed to novobiocin these cells are less viable and have severe chromosome segregation defects. These data suggest that Topo III plays a role in orderly and efficient chromosome segregation.
Rederivation of the parE10 and parC1215 mutations in W3110
As will be obvious below, some of our results deviate significantly from those reported by Lopez et al. (2005). To eliminate the possibility that these differences could be the result of different suppressors acquired in the W3110parE10 and C600parC1215 (EJ182) strains originally isolated by Kato (Kato et al., 1988; 1990) over the years in different laboratories, we engineered both the parE10 mutation (C316Y) and the parC1215 mutation (G725D) into a fresh W3110 background. The resultant W3110parC1215 (CL31) strain was barely viable even at 25°C and could not be used for any analyses. The W3110parE10 (CL001) strain used in this report is the newly rederived strain.
Topo III is required for γ-mediated rescue of parE10 at the restrictive temperature
The isolation of high-copy suppressors of the par and growth defects of ts Topo IV mutations suggested that these suppressor genes either stabilized the ts Topo IV, formed a new topoisomerase, or either enhanced activity or activated one of the other topoisomerases in the cell. The first two possibilities have been ruled out in previous studies (Levine and Marians, 1998; Nurse et al., 2003; Levine, 2000; H. Hassing and K.J. Marians, unpubl. data). Because Topo III was one of the suppressors isolated, we considered the possibility that it played a role in the suppression by the other suppressor genes identified. To test this possibility, we constructed (Table S1) the W3110parE10ΔtopB::kan (CL002) and C600parC1215ΔtopB::kan (BP079) strains and their respective isogenic parents, W3110ΔtopB::kan (BP093) and C600ΔtopB::kan (BP105) and transformed W3110parE10ΔtopB::kan and W3110ΔtopB::kan with the IPTG-regulated overexpression plasmids pLEX5BA-dnaX(γ), which produces only the truncated γ gene product, and pLEX5BA-topB (pBP42).
Overexpression of γ in W3110parE10ΔtopB::kan failed to bypass the growth impediment at 37°C and 42°C under any growth condition tested (even slow-growth conditions on nutrient-limited media) (Fig. 1A). Overexpression of SetB and τ also failed to suppress the growth defect displayed by W3110parE10ΔtopB::kan at 37°C and 42°C (data not shown). As expected, overexpression of topB suppresses lethality of the W3110parE10ΔtopB::kan mutant at 42°C (Fig. 2). Because of the severe growth defect of C600parC1215ΔtopB::kan, discussed below, we were unable to perform similar suppression tests on this mutant. These results imply that the mechanism of suppression of the Topo IV ts mutations by dnaX(γ) and possibly the other suppressors is either through direct stimulation of Topo III or that Topo III provides either a supporting or backup function to Topo IV during chromosome decatenation. As to the former possibility, however, we have been unable to detect any stimulation of Topo III activity by either DnaX or DnaN in our assays in vitro (Levine, 2000 and data not shown).
The catalytic activity of Topo III is required for suppression
To determine if the suppression of the parE10 mutation at non-permissive temperatures is specific to Topo III, we tested whether the other type I topoisomerase present in E. coli, Topo I, could also rescue W3110parE10 at 42°C when overproduced. This proved not to be the case. Whereas overexpression of Topo I [from pLEX5BA-topA (pBP59)] was not toxic to either parental strain (Fig. S2), it could not rescue growth of either W3110parE10 or C600parC1215 at 42°C (Fig. 3). Therefore, Topo III is the only topoisomerase capable of suppressing a Topo IV deficiency efficiently. [Overexpression of DNA gyrase results in incomplete suppression (Kato et al., 1992).]
If Topo III was stimulating Topo IV directly under suppression conditions, one would expect that a catalytically inactive topB allele would work as a suppressor. Using targeted mutagenesis, we replaced the catalytic Tyr with Phe (topBY328F) (Zhang et al., 1995; Harmon et al., 1999) in the topB ORF in pLEX5BA-topB, generating pLEX5BA-topBY328F (pBP47). Unlike wild-type Topo III, overexpression of this inactive mutant of Topo III (Topo III Y328F) did not enable either W3110parE10 or C600parC1215 to grow at 42°C (Fig. 3). [The level of expression of both the wild-type and mutant Topo III was identical (Fig. S3).] These data suggest that the enzymatic activity of Topo III, likely the decatenase function, is important for chromosome segregation under suppression conditions.
Topo III and γ can suppress the parE10 mutation in the absence of either RecQ or RecA
Because of the functional relationship between Topo III and RecQ, and a possible role for Topo III in recombination, we explored a potential function for recQ and recA in the Topo III-dependent rescue of W3110parE10 growth at 42°C. We showed previously that recQ was not required for rescue of W3110parE10 by overexpression of topB (Fig. 4A and Nurse et al., 2003). However, it was possible that the high levels of Topo III present under those conditions could have obscured a recQ requirement. Therefore, because topB is essential for rescue of W3110parE10 by the other suppressors, we asked if either recQ or recA were necessary for topB-dependent suppression of W3110parE10 temperature sensitivity by overexpression of dnaX(γ). Interestingly, we found that overexpression of dnaX(γ) could bypass the Topo IV defect in cells lacking either recQ (Fig. 1A), suggesting that concerted action of RecQ and Topo III is not required for suppression, or recA (Fig. 1B), suggesting that recombination in general was not required for suppression. Similarly, a disruption in recA did not affect the ability of overproduced Topo III to suppress the growth defect of W3110parE10 at 42°C (Fig. 4), suggesting that RecA-dependent recombination is not required for rescue of W3110parE10 at restrictive temperatures by Topo III. Interestingly, removal of RecQ from W3110parE10 made rescue by γ overexpression more efficient at 42°C (Fig. 1A). The reason for this effect is unclear.
Deletion of topB in Topo IV mutants leads to pronounced chromosome segregation defects at permissive temperatures
The inability of the suppressors (excluding topB) to rescue the growth defect of W3110parE10ΔtopB::kan and the failure to detect direct stimulation of Topo III activity by any of the suppressor proteins in vitro, strongly suggested that Topo III was directly involved in chromosome segregation by resolving topological linkages between daughter molecules. We therefore investigated whether Topo III was required for growth of the Topo IV ts mutants at permissive temperatures. In a previous report (Lopez et al., 2005), a synthetic lethal effect between the parE10 and ΔtopB mutations was observed as a failure to obtain at 37°C, bacteriophage P1-mediated co-transduction into W3110parE10 of ΔtopB when a drug marker 50 kbp away from topB was selected, whereas at 30°C the double mutant strain could be recovered. These authors also reported that even at 30°C, the C600parC1215ΔtopB double mutant strain could not be constructed by similar means (Lopez et al., 2005). However, we were able to construct by direct selection both the C600parC1215ΔtopB::kan and W3110parE10ΔtopB::kan double mutant strains by growing all P1 recombinants at 25°C. In agreement with the previous report (Lopez et al., 2005), the parE10 and ΔtopB::kan mutations are synthetically lethal at 37°C (Figs 5 and 6), and the double mutant strain displayed a significant growth impairment at 30°C compared with W3110parE10 (Fig. 5N). In addition, the C600parC1215ΔtopB::kan double mutant strain was severely compromised, failing to grow at 30°C (Fig. 5M) and higher temperatures (data not shown). Ectopic expression of either Topo III or Topo IV from pLEX5BA permits growth of W3110parE10ΔtopB::kan at restrictive temperatures (Figs 2 and S4).
Cellular and nucleoid morphologies of the double mutant strains and their parents were examined. As expected, C600 and C600ΔtopB::kan cells showed no obvious defects (Fig. S5A and B) and had normal cell lengths (Fig. S5G) after 4 h of growth at 25°C. Similarly, C600parC1215 cells also appeared mostly normal, with a small fraction of filamented cells (Fig. 5A and I). Surprisingly, deletion of topB in C600parC1215 resulted in pronounced chromosome segregation defects, even at the favourable temperature of 25°C. These cells exhibited large masses of unsegregated DNA and were filamentous (Fig. 5B and I). Likewise, after 2 h of growth at 30°C, W3110, W3110topB::kan and W3110parE10 cells had normal cell sizes (Fig. S5H and J) and W3110 and W3110ΔtopB::kan cells had normal nucleoid morphology (Fig. S5C and D). The majority of the nucleoids in W3110parE10 cells at 30°C were normal (Fig. 5C). About one-quarter of the cells displayed a phenotype that we refer to as ‘nascent par,’ where an unsegregated mass of DNA was centrally located in a normal-sized cell (Fig. 5C and J). This population of nascent par cells doubled in the W3110parE10ΔtopB::kan double mutant strain. In addition, a significant population of anucleate cells, a phenotype often associated with chromosome segregation defects, was also observed in the double mutant (Fig. 5D and J).
The effect of the ΔtopB mutation could also be observed at the non-permissive temperature of 42°C in the W3110parE10 strain. W3110 and W3110ΔtopB::kan cells grown for 2 h at 30°C and then shifted to 42°C for 1 h appeared wild-type (Fig. S5E and F) with average cell lengths (Fig. S5I and J). Although the par phenotype was obviously present in W3110parE10 cells grown at 42°C, the W3110parE10ΔtopB::kan cells were consistently more filamentous, with the fraction of cells > 15 μm in length increasing by more than twofold (Fig. 5K) and an increased appearance of anucleate cells (Fig. 5E and F). These results indicate that the growth defect of the W3110parE10ΔtopB::kan and C600parC1215ΔtopB::kan mutant strains is linked to a chromosome segregation defect.
To address whether the ΔtopB-mediated synthetic effect on chromosome segregation was specific to the Topo IV ts alleles, we tested for a synthetic relationship between the DNA gyrase ts allele gyrBR136CP171S [from strain N4177, Courts (Oram et al., 1992)] and the ΔtopB mutation. Both the W3110gyrBR136CP171S (BP199) and W3110gyrBR136CP171SΔtopB::kan (BP275) strains grew equally well on plates incubated at 30°C and 37°C, and, as anticipated, were less viable at 42°C (Fig. S6D), although the W3110gyrBts strain was slightly more viable than the W3110gyrBtsΔtopB::kan strain at this temperature (Fig. S6D). Both W3110gyrBts and W3110gyrBtsΔtopB::kan cells cultured for 2 h at 30°C showed no obvious cell or nucleoid morphology defects (Fig. S6A–C), whereas, after 1 h at 42°C both strains amassed large unsegregated nucleoids and failed to divide (Fig. 5G, H and L). Although W3110gyrBtsΔtopB::kan cells were slightly longer than W3110gyrBts cells at 42°C (7.9 μm compared with 7.2 μm, Fig. 5L), it was clear that the ΔtopB::kan mutation had, at most, a minor effect on growth and chromosome segregation in the W3110gyrBts mutant strain. Thus, we conclude that the synthetic lethality of Topo IV ts mutations and the ΔtopB::kan mutation is specific to defects in Topo IV and Topo III.
The chromosome segregation defect and synthetic lethality of the parE10ΔtopB::kan double mutation does not arise from the failure to resolve toxic recombination intermediates
The Topo III and RecQ combination is clearly involved in the resolution of toxic recombination intermediates in eukaryotes by the dissolution of double Holliday junctions (Wu and Hickson, 2003). This has prompted others to investigate whether a similar paradigm holds in prokaryotes. For example, Lopez et al. (Lopez et al., 2005) reported that removal of RecQ suppressed the synthetic lethality of the parE10ΔtopB::kan double mutation. Because we found that neither RecQ nor RecA was required for suppression of W3110parE10 by overexpression of dnaX(γ) (Fig. 1), we tested whether the observed DNA segregation block and growth deficiency of the W3110parE10ΔtopB::kan strain could be alleviated by the introduction of either ΔrecQ or recA938::cat, a recA null mutation.
Comparison by microscopy showed that the lack of RecQ did not alleviate the chromosome segregation defect of W3110parE10ΔtopB::kan cells grown at 30°C (Fig. 6B and E). In addition, introduction of a ΔrecQ mutation to the W3110parE10 strain did not phenocopy the effect of introducing ΔtopB to the W3110parE10 strain (Fig. 6A and E). This observation indicates that linkage of Topo III and RecQ activity, as demonstrated to be required to resolve some forms of recombination intermediates (Oakley and Hickson, 2002), is not responsible for the defects in the W3110parE10ΔtopB strain. Similar results were observed when these strains were incubated at 30°C for 2 h and then shifted to 42°C for 1 h (Fig. S7), although the W3110parE10ΔtopB::kanΔrecQ::cat strain (CL015) showed a decrease in the proportion of very long filaments and an increase in the proportion of anucleate cells compared with the W3110parE10ΔtopB::kan strain (Fig. S7B, D and E). Furthermore, the ΔrecQ mutation did not affect the viability of these strains at either 30°C or 37°C and did not rescue the synthetic lethality of the parE10ΔtopB::kan double mutations at the semi-permissive temperature of 37°C (Fig. 6F). Control experiments (Fig. S8) showed that the ΔrecQ mutation did not affect either cell or nucleoid morphology at either 30°C or 42°C or cell viability at either 30°C or 37°C, either in the presence or absence of Topo III in the parental W3110 strain (Fig. S8A, B, E, F and I).
A general decrease in viability for all recA938::cat mutant strains, which is characteristic of mutants of recA (Figs 6G and S8J), was observed. However, the presence of the recA938::cat mutation did not suppress the growth defect (Fig. 6G) and exacerbated the chromosome segregation defect of W3110parE10ΔtopB::kan, with a dramatic increase in the population of anucleate cells and the appearance of a population of cells in which the nucleoid had become fragmented (Fig. 6C, D, E and G, and Fig. S7C, F and G). The lack of a requirement for either RecQ or RecA for the parE10ΔtopB synthetic lethality argues strongly that the phenotype arises from a chromosome segregation defect and not the failure to resolve toxic recombination intermediates. Given that we rederived the W3110parE strain, it is possible that the relief of synthetic lethality between the parE10 and ΔtopB mutations afforded by mutations in recQ and recA observed by Lopez et al. (2005) is a result of unknown mutations in their strains. Indeed, our original lab stock of W3110parE10 filaments more extensively than the rederived strain at 42°C.
Topoisomerase III mutants are sensitive to novobiocin
Our data supports the notion that Topo III provides either a supportive or backup role to Topo IV in the resolution of catenated daughter chromosomes. If so, then mutants of topB should be hypersensitive to drugs that block chromosome segregation and/or Topo IV. We therefore tested if topB mutants were sensitive to novobiocin, which targets the type II topoisomerases in E. coli (Gellert et al., 1976b; Mizuuchi et al., 1978; Levine et al., 1998). This proved to be the case.
The W3110ΔtopB::kan mutant strain was approximately four times more sensitive to novobiocin than topB+ strains. The wild-type W3110 strain had a MIC50 of approximately 200 μg ml−1 of novobiocin, whereas the W3110ΔtopB::kan strain had a MIC50 of nearly 50 μg ml−1. In fact, the W3110ΔtopB::kan mutant strain failed to grow in the presence of ≥ 100 μg ml−1 of novobiocin (Fig. 7C). In control experiments using a superhelical DNA relaxation assay (Fig. S9), we confirmed that Topo III is unaffected by novobiocin (Srivenugopal et al., 1984).
To determine which type II topoisomerase was mediating the observed increased sensitivity to novobiocin, we examined drug response in the presence of a novobiocin-resistant allele of the GyrB subunit of DNA gyrase (the W3110gyrBts strain (BP199) used in Fig. 5 is also novobiocin resistant). As expected, the W3110gyrBts strain was overall more resistant to novobiocin (MIC50 of ∼ 750 μg ml−1), but the W3110gyrBtsΔtopB::kan mutant was still nearly twofold more sensitive to the drug (MIC50 of ∼ 400 μg ml−1) (Fig. 7D). Furthermore, overexpression of topB in the W3110gyrBts strain conferred a growth advantage at high concentrations of novobiocin (Fig. S10). These data indicate that an increased demand for Topo IV was the cause of the increased sensitivity to novobiocin conferred by the ΔtopB allele and that overexpression of topB in the W3110gyrBts strain relieved demand for Topo IV activity. If this is the case, we would expect to observe chromosome segregation defects in the W3110gyrBtsΔtopB::kan strain in the presence of moderate levels of the drug.
After a 1.5 h incubation with 300 μg ml−1 of novobiocin, W3110gyrBtsΔtopB::kan cells were noticeably more filamentous (average cell length 6.5 μm) compared with W3110gyrBts cells (average cell length 5.0 μm) (Fig. 7A, B and E). Additionally, W3110gyrBtsΔtopB::kan cells had accumulated larger unsegregated nucleoid masses (average area 3.1 μm2) compared with W3110gyrBts cells (average area of 1.3 μm2) (Fig. 7A, B and F), indicating that novobiocin causes a significant block in chromosome segregation in topB mutant strains.
If the observed novobiocin sensitivity of the topB mutant strains was indeed a result of increased challenge for Topo IV, then overexpression of Topo IV should result in bypass of this sensitivity. This proved to be the case. As expected, W3110ΔtopB::kan and W3110gyrBtsΔtopB::kan strains failed to grow as well as either the wild-type or gyrBts strains on novobiocin plates (Fig. 7G). However, overexpression of either Topo III or Topo IV allowed all ΔtopB::kan mutant strains to grow (Fig. 7H). Hence, expression of Topo III in trans can complement the ΔtopB::kan mutation for novobiocin hypersensitivity, as expected, while overproduction of Topo IV can bypass topB-mediated novobiocin sensitivity, suggesting that Topo III is important for chromosome segregation, and its requirement can be bypassed by producing more Topo IV.
Topo III suppresses a Topo IV deficiency by decatenating chromosomes
To date, there has been no observed topB mutant phenotype in prokaryotes that has led to the elucidation of the function of Topo III in the cell. Our previously identified high-copy suppressors that rescue the temperature sensitivity of parE and parC mutant strains (Levine and Marians, 1998; Nurse et al., 2003; Espeli et al., 2003b) have allowed us to dissect the role of Topo III in the cell. The topB gene was essential for the other previously identified high-copy suppressors, including dnaX(γ) (Fig. 1A), dnaX(τ) and setB to rescue the W3110parE10 strain at restrictive temperatures. Based on these findings, we thought that the mechanism of suppression by the high-copy suppressors could be through a direct stimulation of the enzymatic activities of either Topo IV or Topo III. Although we have conducted numerous biochemical and protein-protein interaction studies to test this theory, we have been unable to support this idea. Alternatively, it was possible that the high-copy suppressors (with the exception of topB) worked in an independent fashion, as suggested by others (Grainge et al., 2007), and that Topo III acted in a supportive capacity to Topo IV by actively decatenating chromosomes.
To further support the notion that the decatenation activity of Topo III was essential for chromosome segregation and for the rescue of the W3110parE10 strain at restrictive temperatures, we analysed the requirement for the catalytic activity of Topo III for suppression of the W3110parE10 growth defect at non-permissive temperatures. We report that Topo III is the only topoisomerase in the cell that can efficiently rescue either W3110parE10 or C600parC1215 at the restrictive temperature. Overexpression of DNA gyrase has been shown to only partially rescue temperature-sensitive mutations of parE and parC (Kato et al., 1992), and we show here that overproduction of Topo I completely failed to rescue W3110parE10 at 42°C. In addition, a catalytically dead mutant of topB, when overproduced, is unable to support suppression of either the parE10 or parC1215 mutations at 42°C, suggesting that Topo III's decatenation function is indeed critical for rescue of the W3110parE10 and C600parC1215 strains at non-permissive temperatures.
Even at permissive temperatures, in the absence of topB, W3110parE10 and C600parC1215 were not only compromised for viability, compared with their topB+ counterparts, but had pronounced chromosome and cell division defects consistent with a severe par phenotype. The severity of the defect is emphasized by the fact that even when W3110parE10ΔtopB::kan cells are grown at 30°C, a significant proportion (average of 24%) of the cells in the population die, whereas mortality frequencies under the same growth conditions for W3110parE10 were < 1%. Moreover, the par phenotype displayed by the C600parC1215ΔtopB::kan mutant strain was consistently more severe than that of W3110parE10ΔtopB::kan, suggesting that the par phenotype in topB mutant strains worsen with increasing loss of Topo IV activity. These observations indicate that the well-studied W3110parE10 and C600parC1215 strains depend on Topo III for survival. Even when these parE and parC ts mutant strains are grown 30°C, the Topo IV enzyme produced in either W3110parE10 and C600parC1215 mutant strains is inadequate to carry out efficient decatenation reactions for the timely DNA segregation that is critical for productive cell division and growth. Although the chromosomal levels of Topo III are significantly lower than that of the other DNA topoisomerases (DiGate and Marians, 1989), Topo III's strong decatenase activity (DiGate and Marians, 1988) is likely the key to its ability to partially substitute for a Topo IV defect in the cell.
The topB mutant phenotype is not the result of failure to resolve recombination intermediates
Numerous studies have shown biochemical and genetic associations between Topo III, RecQ, and cellular processes such as the resolution of converging replication fork complexes (Suski and Marians, 2008) and the dissolution of double Holliday junctions (Mankouri and Hickson, 2007). Using our suppressor-based genetic assay, we demonstrated that recQ was not required for either Topo III or γ (when in excess) to rescue the W3110parE10 strain. Rescue of W3110parE10ΔrecQ (CL004) by γ was Topo III-dependent, demonstrating that RecQ was not required for endogenous levels of Topo III to suppress the Topo IV deficiency and for successful DNA segregation. Consistent with these findings, there were no compounded effects on viability or chromosome segregation in W3110parE10 strains with ΔrecQ mutations at either permissive or non-permissive temperatures. Therefore, Topo III does not require its cognate helicase to unlink daughter chromosomes and to rescue W3110parE10, and the genetic evidence suggests that its substrate in vivo could be precatenanes.
Previous observations (Lopez et al., 2005) have suggested that the synthetic effect on viability of the parE10ΔtopB and parC1215ΔtopB double mutations was because of the accumulation of recombination intermediates, and that either introducing a recQ mutation or abolishing recombination (with a recA mutation) could suppress the described synthetic lethality. The authors used their observations to support a role for Topo III in recombinational repair of DNA lesions. This model is inconsistent with our findings.
Based on our biochemical assays, suppression of W3110parE10 and C600parC1215 temperature sensitivity by overexpression of dnaX(γ) and setB is unlikely to occur by direct stimulation of Topo III, we would then expect overexpression of dnaX(γ) or the other high-copy suppressors to suppress the formation of such toxic recombination intermediates in W3110parE10ΔtopB. Thus, failure of overexpression of dnaX(γ) to rescue W3110parE10ΔtopB is consistent with a role in chromosome segregation for Topo III. Of course, it is also possible that the high-copy suppressors potentiate Topo III-catalysed decatenation of the daughter chromosomes in a manner that is undetected by our biochemical assays. However, we did not observe any differences in either growth or state of the nucleoids between W3110parE10ΔtopB or W3110parE10ΔtopBΔrecQ: both strains failed to grow at semi-permissive temperatures and to segregate chromosomes properly. Overexpression of dnaX(γ) also failed to suppress the parE10ΔtopBΔrecQ triple mutation combination at either 37°C or 42°C. In addition, the pronounced chromosome segregation defects in the W3110parE10ΔtopB and C600parC1215ΔtopB mutant strains can be reversed by overexpression of Topo IV.
Finally, abolishing recA-dependent recombination had no effect on the described parE10ΔtopB phenotype of growth, rescue by high-copy suppressors, or chromosome segregation. RecA was not required for the parE10ΔtopB phenotype, and introduction of a recA null mutation neither permitted growth of W3110parE10ΔtopB nor alleviated the chromosome segregation block. Therefore, we conclude that the parE10ΔtopB synthetic lethality arises from a chromosome segregation problem, rather than because of the accumulation of toxic recombination intermediates.
Topo III participates in chromosome segregation
The findings in this study provide strong evidence that Topo III participates in chromosome segregation. To address whether Topo III was essential for chromosome segregation, we asked if topB mutants were more susceptible to agents that target Topo IV and block chromosome segregation. Mutants of topB treated with novobiocin were unable to grow in the presence of novobiocin at concentrations tolerated by topB+ strains, and these novobiocin-treated cells displayed aberrant chromosome segregation. This finding suggests that topB mutants are novobiocin sensitive because they cannot properly segregate their chromosomes in the presence of drug. The novobiocin sensitivity of the W3110ΔtopB mutant strain is the first identified phenotype of topB mutations that supports a clear role and requirement for Topo III in chromosome segregation when Topo IV activity is reduced, as herein by treatment with novobiocin. Furthermore, these observations are consistent with the phenotype displayed by the parE10ΔtopB double mutation. We consider it unlikely that enervation of Topo IV creates a new activity for Topo III and therefore suggest that the activities of both Topo IV and Topo III are necessary for segregation of the chromosomes in E. coli. The novobiocin sensitivity of the topB mutation is specific to a defect in chromosome segregation because overexpression of either Topo III or Topo IV can alleviate the novobiocin sensitivity of topB mutant strains, suggesting that an excess of Topo IV can suppress the requirement for Topo III. Moreover, the introduction of novobiocin-resistant alleles of gyrB did not suppress the novobiocin sensitivity of topB mutant strains.
Because Topo III rescues W3110parE10 without the assistance of RecQ and in the absence of recombination, and requires single-stranded DNA gaps to decatenate, it is probable that its mechanism of action occurs during DNA replication by resolving precatenanes (Fig. 8). Therefore, we envision the action of three topoisomerases as being necessary during DNA replication to ensure complete topological disengagement of the parental strands: DNA gyrase acts ahead of the replication fork, removing positive supercoils. Any supercoils that diffuse behind the fork, leading to the formation of precatenanes, are resolved by Topo III prior to ligation of the Okazaki fragments. And catenanes that do form are resolved by Topo IV.
Because Topo IV and Topo III are the main decatenating enzymes, it is clear why the synthetic effect of the topB mutation on viability and chromosome segregation is specific to the parE and parC ts alleles. Combining the gyrB ts allele with a topB deletion mutation has only minor consequences for the cell because Topo IV is available to resolve the topological challenges. For decatenation, it is clear that Topo IV is the workhorse of the cell, because its function cannot be completely replaced by either DNA gyrase or Topo III. This could either be because of a persistent burden of catenanes that is too great of a challenge for either Topo III or DNA gyrase, or an additional essential role for Topo IV in other unrelated functions.
In consideration of our previous findings that Topo IV activity is temporally regulated and functions specifically at the later end stages of DNA replication (Espeli et al., 2003a), one could predict that W3110ΔtopB mutant strains should have a delay in chromosome segregation. However, the interplay between Topo IV and Topo III and factors governing their regulation has not been explored. It is possible that the cell regulates the activities of Topo III and Topo IV with respect to the demand for decatenation as it does the activities of DNA gyrase and Topo I to ensure the proper superhelical state of the DNA (Menzel and Gellert, 1983). Interestingly, reduced gene expression of parE in Staphylococcus aureus leads to an increase in topB transcript levels (Ince and Hooper, 2003).
Bacterial strains and growth conditions
Escherichia coli strains are described in Table S1 and were cultured in either LB, Minimal A (Min A), or M9 medium supplemented with thiamine (40 μg ml−1), threonine (40 μg ml−1) and leucine (40 μg ml−1). The following concentrations of antibiotics were used: ampicillin (Ap), 100 μg ml−1; carbenicillin (Cb), 50 μg ml−1; kanamycin (Km), 50 μg ml−1; chloramphenicol (Cm), 34 μg ml−1; novobiocin (Nov), 600 μg ml−1. Unless indicated otherwise, wild-type strains were incubated at 37°C, whereas strains with ts mutations were incubated at 30°C. W3110 is the parental strain of W3110parE10 (Kato et al., 1990) and C600 is the parental strain of C600parC1215 (EJ812) (Kato et al., 1988).
Plasmids (Table S2) were constructed by standard molecular genetic procedures. All plasmid preparations were done using Miniprep or Midiprep Spin Kits (Qiagen). DNA manipulations with restriction endonucleases and bacteriophage T4 DNA ligase were done according to the manufacturer's (NEB) recommendations. PCR amplifications were done with high fidelity ExTaq polymerase (TaKaRa), KOD HiFi polymerase (Novagen), or Phusion DNA polymerase (NEB) for 30 cycles, and all primers are listed in Table S3. To clone specific genes, either C600 or W3110 genomic DNA was used as template for PCR. The QiaexII kit (Qiagen) was used for agarose gel extraction. PCR products and constructed plasmids were confirmed by nucleotide sequencing at the MSKCC DNA Sequencing Facility.
Rederivation of the parE10 and parC1215 mutations in W3110
Nucleotides 3171470–3173470 were amplified from genomic DNA prepared from our original lab stock of W3110parE10 using primers that introduced BglIII and SalI restriction enzyme sites at the 5′ and 3′ ends of the amplified DNA fragment respectively (parE10top and parE10bot, Table S3). This DNA fragment had 1 kbp of chromosomal DNA flanking either side of the parE10 mutation. This DNA fragment was digested with these restriction enzymes and ligated to similarly digested pKO3 DNA (Link et al., 1997) to give pKO3-parE10.
The parE ORF was amplified from genomic DNA prepared from W3110 using primers that introduced EcoRI and HindIII restriction enzyme sites to the 5′ and 3′ ends of the amplified DNA fragment respectively (parEtop and parEbot, Table S3). This DNA fragment was digested with these restriction enzymes and ligated to similarly digested pRC7 DNA (de Boer et al., 1989) to give pRC7-parE. This plasmid expresses an IPTG-inducible LacZ–ParE fusion and is lost very quickly from cells in the absence of ampicillin.
W3110 cells were transformed with pKO3-parE by electroporation and selected on LB-Cm plates at 30°C. A single transformant was grown in LB-Cm medium at 30°C until mid-log, serially diluted in LB, plated on pre-warmed LB-Cm plates, and grown at 43°C. A single co-integrate was picked from this plate, grown to mid-log and maintained in LB-Cm at 43°C. These cells were transformed with pRC7-parE and plated on LB-Cm-Ap plates at 43°C. Colonies confirmed to have both plasmids established were then grown on LB plates supplemented with Cm, Ap, and 100 μM IPTG to induce expression of the LacZ–ParE fusion protein. Several of the resulting colonies were dispersed into 1 ml of LB medium and cells were streaked on LB plates supplemented with Ap, 100 μM IPTG, and 5% sucrose and grown at 30°C. Clones were first checked for loss of pKO3-parE10 by replica plating onto LB-Cm-Ap plates at 30°C. Ap-resistant, Cm-sensitive clones were then replica plated onto LB-IPTG plates at 30°C to check for loss of pRC7-parE. The resulting clones were checked for temperature sensitivity, as well as ensuring the loss of pRC7-parE, by growth on LB plates at 43°C.
For the parC1215 mutation the methodology was the same except that pKO3-parC1215 carried nucleotides 3160813–3162822 amplified from chromosomal DNA of C600parC1215 (EJ812) and pRC7-parC carried the ParC ORF amplified from W3110 genomic DNA and the primers used to introduce the ParC ORF to pRC7 added SalI and HindIII restriction sites to the PCR product.
Construction of the TB28ΔtopB::FRTkan mutant
The ΔtopB::FRTkan mutation was constructed by linear replacement (Datsenko and Wanner, 2000). FRT sites flank the ΔtopB::kan cassette and successful recombination of the allele resulted in the replacement of the topB ORF. The primers DtopB-1 and DtopB-2 (Table S3) were used to PCR amplify the ΔtopB::FRTkan cassette. The PCR fragments were transformed into TB28 as described previously (Datsenko and Wanner, 2000) and recombinants were selected in LB supplemented with Km. The ΔtopB::FRTkan mutation was verified by PCR and DNA sequencing.
Bacteriophage P1 transduction of mutant alleles
The following strains:TB28ΔtopB::FRTkan, CRL31(ΔrecQ::FRTcat), SS391 (recA938::cat) and N4177 (gyrBR136C P171S; Courts) served as P1 donors for the transduction of the ΔtopB::FRTkan, ΔrecQ::FRTcat, recA938::cat and gyrBts alleles into strains described in Table S1.
Overnight cultures grown in LB were diluted to an OD600 of 0.005 in LB medium and grown at the indicated temperatures. At the indicated times, cells were harvested by centrifugation at 1500 g for 10 min. Pellets were resuspended in 1× PBS. To stain the cell membranes and DNA, FM4-64FX (Molecular Probes) at 1 μg ml−1 and DAPI (Molecular Probes) at 1 μg ml−1, respectively, were added to the cell suspension. The suspensions were spread onto slides coated with 0.1% poly-l-lysine (Sigma). Cells were fixed by overlaying the slides with 1 ml of 2.5% paraformaldehyde, 0.03% glutaraldehyde, 1× PBS for 30 min. Slides were washed three times with 1× PBS and allowed to dry at room temperature. The cells were visualized with a Nikon Eclipse Ti fluorescence microscope. Nikon Elements software was used to determine cell length and nucleoid area; a minimum of 300 cells and 500 nucleoids, respectively, were measured.
Strains were grown overnight in LB supplemented with Ap at 30°C. The saturated cultures were then serially diluted and spotted (10 μl) on either LB or Min A plates containing Ap and 100 μM IPTG. Plates were incubated at 30°C and 37°C and/or 42°C.
Novobiocin sensitivity plating assays
Cultures were grown overnight in LB at 37°C for W3110 strains and 30°C for gyrBtsNov (BP199) strains. The cultures were diluted to an OD600 of 0.001 in fresh LB, grown for 1 h at the appropriate temperature, and plated on LB plates containing varying concentrations of novobiocin. W3110 strains were incubated at 37°C overnight, whereas gyrBtsNov strains were incubated at 30°C for approximately 30 h. Survival was determined by the following: average colony-forming units on novobiocin plates/average colony-forming units on LB only plates. MIC50 is defined as the minimum inhibitory concentration: The concentration of drug that is required to inhibit 50% of growth.
Topo III and Topo IV bypass of novobiocin sensitivity
W3110, W3110ΔtopB::kan (BP093), W3110gyrBtsNov (BP199) and W3110gyrBtsNovΔtopB::kan (BP275) were transformed with pLEX5BA, pLEX5BA-parEparC and pLEX5BA-topB (pBP42). Transformants were grown and diluted back as described above. Prior to plating, strains were grown in LB with 100 μM IPTG at 37°C (for W3110 strains) or 30°C (gyrBtsNov strains) for 2 h Cultures were titered and spotted on LB plates supplemented with 100 μM IPTG and novobiocin (50 μg ml−1 for W3110 strains and 300 μg ml−1 for gyrBtsNov strains).
We thank Olivier Espeli, John Petrini and Rod Rothstein for comments on the manuscript, and Catherine Suski-Grabowski for strains. These studies were supported by NIH grant GM34558.