A Gyrase Mutant with Low Activity Disrupts Supercoiling at the Replication Terminus

When a mutation in an essential gene shows a temperature-sensitivephenotype, one usually assumes that the protein is inactiveat nonpermissive temperature. DNA gyrase is an essential bacterialenzyme composed of two subunits, GyrA and GyrB. The gyrB652mutation results from a single base change that substitutesa serine residue for arginine 436 (R436-S) in the GyrB protein.At 42°C, strains with the gyrB652 allele stop DNA replication,and at 37°C, such strains grow but have RecA-dependent SOSinduction and show constitutive RecBCD-dependent DNA degradation.Surprisingly, the GyrB652 protein is not inactive at 42°Cin vivo or in vitro and it doesn't directly produce breaks inchromosomal DNA. Rather, this mutant has a low k_cat comparedto wild-type GyrB subunit. With more than twice the normal meannumber of supercoil domains, this gyrase hypomorph is proneto fork collapse and topological chaos near the terminus ofDNA replication.

INTRODUCTION

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Introduction

Temperature-sensitive (TS) mutants have been used to study complexbiochemical pathways in prokaryotic and eukaryotic organismsalike. By comparing phenotypes of a TS mutant at permissiveand restrictive temperatures, important insights can be obtainedabout the progression of intermediates through a biochemicalpathway. For example, TS mutants guided early studies of DNAreplication and provided the initial evidence for three differentcellular polymerases (28). TS mutants also showed that replicationinvolves numerous genes, and a large mutant collection guidedthe in vitro reconstruction of replication initiation and elongation(44, 72). In bacteriophages ${lambda}$ and Mu, TS repressors were foundthat undergo a change in protein conformation causing repressor-DNAcomplexes to dissociate at nonpermissive temperatures (24, 31,69). Physiological studies after shifts from permissive to nonpermissivetemperature revealed relevant promoters and important biochemicalsteps necessary for the onset of the lytic cycle in phage development(19, 30). Thus, it has become expected that when a gene confersa TS phenotype, the cause is a temperature-dependent changein the mutant protein's structure or catalytic activity.

DNA gyrase has a unique and essential role in prokaryotic replicationand transcription. The enzyme catalyzes the introduction ofnegative supercoils into covalently closed DNA molecules atthe expense of ATP binding and hydrolysis. The enzyme is essentialin most prokaryotes and is a critical target for antimicrobialchemotherapy. The quinolone antibiotics are among the most potentantibacterial agents known, and they kill bacteria by turningDNA gyrase (and the closely related enzyme topoisomerase IV[Topo IV]) into a DNA-reactive cytotoxin (23).

Gyrase plays at least two essential roles in chromosome structureand function. First, it condenses DNA by converting relaxedDNA molecules into a dynamic, underwound, and plectonemicallysupercoiled state (12). DNA in living cells is compartmentalizedinto subchromosomal regions called domains, and the dynamicsuperhelical structure within a domain affects many processeslike transcription, recombination, site-specific recombination,and transposition (12, 18, 33, 36). Second, to unwind the Watsonand Crick strands during DNA synthesis, a cell must remove 400,000positive coils in each chromosome-equivalent of DNA and resolveall catenane links between the replicated chromosomes. Gyrase-drivennegative supercoils facilitate both the unwinding and the TopoIV-dependent unlinking of parental strands during bacterialDNA replication (54, 70).

We previously described TS mutants of gyrase with an increasednumber of superhelical domains. At the permissive temperature(30°C), mutations in gyrB were found to cause more thantwice as many domains as wild-type (WT) strains over a 100-kbinterval spanning the his and cob operons (64, 66). The gyrB652mutation was originally isolated by Gari et al. (27), and itcauses induction of the SOS response at 37°C and shows syntheticlethality in combination with recA or recB mutations. Here,we isolated GyrB652 gyrase and compared it to the WT enzyme.To our surprise, the GyrB652 gyrase was not TS in vitro or invivo. The enzyme had a low turnover number (k_cat) in vitro,and in vivo it caused a dramatic change in supercoil dynamicsnear the replication terminus.

MATERIALS AND METHODS

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Materials and Methods

Strains and cloning. Bacterial strains used in this work are listed in Table 1. Boththe WT gyrB and the gyrB652 gene from Salmonella enterica serovarTyphimurium were cloned into pET21(a) in Escherichia coli DH5 ${alpha}$ .Then, plasmids were moved into E. coli BL121:: ${lambda}$ DE3 (67) for proteinexpression under control of the phage T7 RNA polymerase. PlasmidpRC06 contains the gyrB gene from WT Salmonella strain LT2,and pRC04 contains the gyrB652 gene with an A-C transversionthat changes the Arg 436 codon to Ser.

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TABLE 1. Strains used^a

Enzyme purification. WT and mutant GyrB proteins were expressed and purified usingthe same protocol. Five milliliters of an overnight culturewas used to inoculate 500 ml of Luria broth (LB) containing50 µg/ml ampicillin. The culture was agitated at 30°C,and cell density was monitored hourly. When the culture reachedan optical density at 650 nm of 0.85, IPTG (isopropyl-ß-D-thiogalactopyranoside)was added to a final concentration of 0.1 mM, and incubationat 30°C was continued for 2 h. Cultures were chilled to4°C, and the cells were harvested by centrifugation andresuspended in 60 ml of TGED buffer (50 mM Tris-HCl, pH 8.0,10% glycerol, 1 mM EDTA, 1 mM dithiothreitol [DTT]). Lysis wasinduced by addition of 20x-concentrated lysis buffer which madethe solution (at final concentration) 2 mM DTT, 20 mM EDTA,100 mM KCl, 0.2% Brij 58, and 0.4 mg/ml egg white lysozyme.The mixture was placed in centrifugation tubes, incubated at4°C for 30 min, and centrifuged at 35,000 rpm in a Beckmantype 60 Ti ultracentrifuge rotor for 1 h at 4°C. Solid streptomycinsulfate was added to the supernatant at a final concentrationof 4%, and the mixture was stirred at 4°C for 30 min. Aftercentrifugation for 30 min in a Beckman J21 rotor at 7,000 rpm,solid ammonium sulfate was added to the supernatant at a finalconcentration of 50% saturation. After centrifugation in a BeckmanJ21 rotor at 7,000 rpm, the pellet was dissolved in 1x TGEDbuffer with 1 M NaCl. Dialysis was carried out overnight inTGED with 1 M NaCl.

Novobiocin was covalently coupled to CNBr-activated Sepharose4B according to instructions supplied by Amersham Biotech. Tenmilliliters of a dialyzed protein solution was applied to a5-ml (bed volume) novobiocin-agarose column, and the appliedprotein solution was recirculated through on the column for1 h. Washing with 15 ml of solution containing 2 M urea in 1xTGED eluted most of the protein bound to the resin. GyrB proteinwas then eluted in 6 M guanidine-HCl in 1x TGED. Fractions withthe highest protein concentration were identified by Bio-Radprotein assays and dialyzed for 12 h against 1x TGED at a maximumprotein concentration of 1 mg/ml, which allowed the refoldingof the purified GyrB protein. The dialyzed solution was clarifiedby centrifugation, and the supernatant was assayed for supercoilingactivity after addition of GyrA protein. Renatured GyrB proteinwas applied to a 5-ml DEAE column equilibrated with 1x TGEDbuffer. Active GyrB protein eluted in a 100-ml TGED gradientthat varied linearly from 0 to 0.5 M NaCl. Fractions with thehighest specific activity were identified using GyrA-complementedDNA supercoiling assays (35). Active fractions were pooled andapplied to a 200-ml Sephacryl S-200 column equilibrated in 0.2M KPO4, pH 7.4, 10% glycerol, 1 mM EDTA, and 5 mM DTT. Sephacrylfractions (20 ml) near the void volume were assayed and concentratedby dialysis into 50% glycerol containing 10 mM Tris-HCl, pH7.4, 50 mM KPO4, 1 mM DTT, and 0.1 mM EDTA, which reduced thevolume by half. Both the WT and GyrB652 subunits were stableat –20°C in this buffer.

Genetic methods. PCR products were "sewn" into a 9-kb module containing a completeWT Lac operon (lacIZYA), the gentamicin resistance gene aacC1[aminoglycoside-(3)-N-acetyltransferase] from Tn1696 flankedby a pair of res sites, and a pair of FLP recognition target(FRT) sites. The PCR product of the 9-kb module was cloned intoplasmid pCR-XL-TOPO (Invitrogen) between nucleotides 336 and337, generating the plasmid pPM1 (sequence available on request).

To introduce a Lac-Gen-res module into a chromosome, a FRT sitewas first inserted into a specific location in the Salmonellachromosome using techniques and reagents described by Datsenkoand Wanner (14). Strains containing a single chromosomal FRTsite and the pCP20 plasmid, which expresses FLP recombinaseat 42°C, were grown to late log phase at 30°C and incubatedat 42°C for 10 min to induce FLP expression. After chillingin ice water, cells were made competent by 30 s of centrifugationand resuspension in ice-cold water and were electroporated withplasmid pPM1. FLP recombinase catalyzes the excision of a FRTcircle from pPM1 and its reintegration into the chromosomalFRT site. Each culture was infected with P22 at low multiplicityand incubated at 30°C in a shaking incubator until celllysis (37). Strains NH3514 (LT2/pJBREScI) or NH3515 (gyrB652/pJBREScI)were transduced with the resulting P22 phage to generate strainsNH3504 to -3511. Gentamicin-resistant transductants that stainedblue on X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)plates and were Kan sensitive were subsequently tested for thecorrect location of the Lac operon using primers in the bacterialchromosome and in the Lac-Gen-res module.

RESULTS

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Results

The GyrB652 protein is not TS for supercoiling. Gari et al. isolated the Salmonella gyrB652 mutant and reportedit to be constitutively induced for SOS functions and to showRecBCD-dependent DNA degradation (27). To examine the biochemicalproperties of this mutant, we purified Salmonella WT GyrB andGyrB652 proteins (see Materials and Methods). When mixed witheither E. coli or Salmonella GyrA protein, the specific activityof WT S. enterica serovar Typhimurium GyrB-containing gyrasewas comparable to purified WT E. coli gyrase— at 10⁵ U/mgof purified protein. The specific activity of gyrase reconstitutedwith the Salmonella GyrB652 protein was 1/3 to 1/5 that of theWT protein reconstituted with either E. coli or Salmonella GyrA.

Gyrase containing the WT Salmonella GyrB or the GyrB652 proteinwas tested in supercoiling assays at three temperatures—30°C,which is permissive for growth; 37°C, which shows an SOSphenotype for GyrB652; and 42°C, which is nonpermissivefor GyrB652. Supercoiling assays are not linear with respectto added enzyme because DNA intermediates become distributedin a family of bands with electrophoretic mobility between relaxedand fully supercoiled positions. A unit of gyrase has been definedby an endpoint dilution assay in which 1 unit of enzyme supercoils0.2 µg of relaxed plasmid DNA to a highly supercoiledband after 30 min at 30°C, as measured by gel electrophoresisin an agarose gel lacking an intercalator (35). The activitiesof different gyrase preparations were compared using either1 U or 4 U of mutant or WT enzyme incubated for half the normaltime (15 min). Gyrase made with the WT GyrB subunit completelysupercoiled the relaxed plasmid in 15 min at 30°C (Fig.1A, lane 2), whereas the GyrB652 enzyme converted the substrateto a family of supercoiled bands that migrated between nickedand fully supercoiled forms (Fig. 1A, lane 1). At 37°C,the WT assay with 1 U of enzyme was slightly less active thanthe corresponding 30°C reaction (Fig. 1A, lane 6), but supercoilingwas well matched to the GyrB652 enzyme (Fig. 1A, lane 5). At42°C, WT enzyme was less active than at 37°C (Fig. 1A,lane 10), but the GyrB652 enzyme again showed activity comparableto WT enzyme (Fig. 1A, lane 9). Reactions containing 4 U ofeither the WT or GyrB652 gyrase completely supercoiled the relaxedplasmid at all temperatures (Fig. 1A, lanes 3, 4, 7, 8, 11,and 12). Thus the GyrB652 enzyme was no more temperature sensitivethan WT gyrase as determined by an in vitro supercoiling assay.

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FIG. 1. Temperature effects on DNA supercoiling in vitro or in vivo. A. Supercoiling assays were assembled with relaxed pMP1000 plasmid DNA and either 1 U (lanes 1, 2, 5, 6, 9, and 10) or 4 U (lanes 3, 4, 7, 8, 11, and 12) of DNA gyrase containing GyrB652 subunits (odd-numbered lanes) or WT GyrB (even-numbered lanes). After 15 min of incubation at 30°C (lanes 1 to 4), 37°C (lanes 5 to 8), or 42°C (lanes 9 to 12), reactions were terminated by addition of SDS and the products were run on a 1% agarose gel. Arrows at the side indicate the position of the relaxed substrate (Rel) and supercoiled product (Sc). Intermediates in the supercoiling reaction run between these two positions. B. Plasmid DNAs (pUC19) isolated from exponential cultures of strain NH2585 (WT; lanes 1 and 2) or NH2589 (GyrB652; lanes 3 and 4) were run in an agarose gel containing Tris-borate-EDTA and 25 µM chloroquine phosphate. In lanes 1 and 3, cells were incubated continuously at 30°C, while in lanes 2 and 4, cells were shifted to 42°C for an hour before harvest. The arrows indicate the centers of the topoisomer distribution.

Supercoiling reactions in vitro might differ from in vivo reactionsin critical ways. For example, at the restrictive temperaturein vivo a mutant enzyme could be refolded by molecular chaperonesor degraded by cellular proteases. Thus, the in vivo supercoilingphenotype for a TS GyrB652 strain might not be apparent in vitro.To test gyrase activity in vivo, the supercoil density of theplasmid pUC19 was measured at permissive and nonpermissive temperatures.Plasmid supercoiling is exquisitely sensitive to change in gyraseactivity (32, 46, 57). For example, inhibition of gyrase withnovobiocin or a fluoroquinolone leads to a loss in plasmid linkingnumber within minutes of drug addition (53, 75). To test invivo supercoiling, a WT and a GyrB652 mutant strain was grownto mid-log phase (optical density at 65 nm, 0.6) at 30°C.A portion of each culture was shifted to 42°C and incubatedfor 1 h. Plasmid DNA was purified from both the 30°C and42°C cultures, and aliquots of plasmid DNA were loaded ontoan agarose gel containing 25 µM chloroquine phosphate,which partially unwinds DNA to reveal the distribution of topoisomers.For Salmonella strain NH2585 (WT), the average superhelicaldensity of plasmids isolated from cells held at 30°C wassimilar to plasmids from cells incubated at 42°C (Fig. 1B,lanes 1 and 2). For strain NH2589 (gyrB652), the 30°C pUC91plasmid topoisomer distribution differs from that for WT cellsgrown at 30°C in two ways (Fig. 1B, lane 3). First, thedistribution is broader. Second, the median of the distributionis shifted one or two topoisomers to the less-supercoiled level(toward the gel bottom in this system). These results agreewith supercoil distributions published previously (64), wherewe found a slightly less supercoiled plasmid in the gyrB652mutant at 30°C. When the NH2585 strain is incubated for1 h at the nonpermissive temperature of 42°C, the pUC19plasmid supercoil distribution remained the same (Fig. 1B, lane4). Treatment of either NH2589 (WT) or NH2585 (gyrB652) withan inhibitory but nonlethal dose of the gyrase inhibitor novobiocincaused a shift to a less-supercoiled band position (data notshown). Thus, the GyrB652 subunit does not cause a significanttemperature-dependent loss of plasmid supercoiling at 42°Cin vivo.

The GyrB652 subunit has low catalytic activity. The in vitro and in vivo supercoiling results (Fig. 1) leadto a dilemma. The mutant enzyme does not behave as a classicTS protein; it shows no loss of activity at the nonpermissivetemperature. Nonetheless, it has a striking TS phenotype. Themeasurable difference in plasmid supercoil density when themutant is compared to WT was not influenced by temperature andthere was no evidence of temperature-dependent gyrase-inducedDNA cleavage in vitro or in vivo (26, 27; R. Chen, unpublisheddata). These observations required a new hypothesis. If thedifference in the specific activity of the WT GyrB and GyrB652preparations is real (i.e., not due to a much larger fractionof misfolded or otherwise inactive protein in the GyrB652 preparation),then low catalytic efficiency, k_cat, might cause the SOS responseand RecBCD-dependent degradation of DNA.

To test the hypothesis, an assay was required that is not biasedby the possible presence of inactive enzyme. Supercoiling assaysdo not distinguish between slow and partially inactive proteinsbecause the reaction is somewhat distributive. Measuring a supercoilrate per enzyme molecule requires knowing the number of activeenzymes, but cleavage assays meet the requirement. A high-affinitybinding site for gyrase in bacteriophage Mu (the strong gyrasesite [SGS]) is bound and cleaved efficiently after additionof fluoroquinolones in vitro and in vivo (50, 60). The SGS ishighly preferred as a gyrase binding site, and only one gyrasetetramer can bind to the SGS. During strand transfer the complexpasses through a covalent intermediate wherein the DNA backboneis linked to tyrosine-122 of the GyrA subunit (11, 43). In theabsence of drugs, covalent gyrase-DNA complexes are a fleetingspecies because biochemical equilibrium strongly favors a noncovalentform of enzyme-bound DNA (34). Enoxacin shifts the equilibriumso that gyrase makes an efficient double-strand break followingthe addition of denaturing agents like sodium dodecyl sulfate(SDS) (60). Because gyrase does not turn over in cleavage assays,DNA breakage of preformed gyrase-DNA complexes monitors onlythe active protein bound to a single site on DNA.

To measure the transition from a bound but uncleaved complexto cleavage complex, WT GyrB- and GyrB652-reconstituted gyrasewas incubated for an hour with plasmid pMP1000, which has a1-kb fragment with the Mu SGS cloned into a pBR322-derived plasmid(50). Digestion with EcoRI generates a 3.69-kb linear molecule,but after gyrase cleavage at the SGS, two bands appear: a 2.78-kbfragment and a small rapidly migrating 0.91-kb band (not seenin Fig. 2). SDS was added at different time intervals afterenoxacin addition to measure the kinetics of cleavage. Thisexperiment eliminates the contribution of inactive GyrB subunitsbecause they don't bind the SGS or cleave DNA.

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FIG. 2. A. Physical map of plasmid pMP1000. The positions of the phage Mu DNA (bold line) and the SGS are indicated along with the position of a single EcoRI restriction site. B. Kinetics of WT gyrase cleavage in the presence of enoxacin. Plasmid pMP1000 was incubated with EcoRI and reconstituted WT gyrase as indicated at 37°C for 1 h. After the addition of enoxacin, aliquots were withdrawn and quenched with SDS and proteinase K at different times. The marker in lane M is a 1-kb DNA ladder; gyrase cleavage at the SGS produces a 2.7-kb band. Incubation with enoxacin was as follows: 0 min, lane 1; 0.5 min, lane 2; 1 min, lane 3; 1.5 min, lane 4; 2 min, lane 5; 3 min, lane 6; 4 min, lane 7; 5 min, lane 8; 10 min, lane 9; and 30 min, lane 10. C. Kinetics of GyrB652 gyrase cleavage in the presence of enoxacin. pMP1000 DNA was incubated as described above with GyrB652 gyrase. Enoxacin was added and incubation continued at 37°C for the following times: 0 min, lane 1; 1 min, lane 2; 2 min, lane 3; 3 min, lane 4; 4 min, lane 5; 5 min, lane 6; 10 min, lane 7; and 30 min, lane 8. The positions of EcoRI-linearized pMP1000 and gyrase-cleaved product are marked as EcoRI and SGS-cut at the right edge of panel C. D. Temperature effects on the kinetics of enoxacin-dependent cleavage. Cleavage reactions were carried out using WT gyrase (open squares) and GyrB652 gyrase (filled squares) at 30°C (left panel), 37°C (central panel), and 42°C (right panel). Cleavage levels were quantitated using Bio-Rad Quantity One video imaging software, and the level is given as a percentage of product formed after 30 min of incubation with enoxacin (see panels B and C). The estimated time to achieve half-maximal cleavage was about 1 min at 30°C, 45 s at 37°C, and 30 s at 42°C for WT gyrase and 5 min at 30°C, 1.6 min at 37°C, and 1 min at 42°C for GyrB652 gyrase.

DNA cleavage was slower for GyrB652 gyrase than for the WT gyrase(Fig. 2B, C, and D). At 30°C, the time required to reachhalf-maximal cleavage of the SGS was 1 min for the wild typeand 3.5 min for the GyrB652 gyrase (Fig. 2D). Measurable differenceswere also found at 37°C and 42°C: wild-type and mutantenzymes reach half-maximal cleavage in 45 s versus 1.5 min at37°C and 30 s versus 1 min at 42°C, respectively. TheQ10 was observed with both forms of gyrase; the cleavage ratesincreased about twofold as assay temperatures rose from 30°Cto 42°C. Nonetheless, gyrase made with the GyrB652 subunitcleaved DNA more slowly than WT at all temperatures tested.These data support a kinetic explanation for the Gyr652 TS phenotyperather than enzyme inactivation at nonpermissive temperature.

Strains with the gyrB652 allele lose supercoil dynamics near the dif site. If gyrase is active at 42°C, why does a GyrB652 strain stopgrowth and DNA synthesis at 42°C rather than just slowingdown? The terminus of replication has unique topological rolesin bacterial chromosome replication and segregation. BidirectionalDNA synthesis in both E. coli and Salmonella leads the two forksthat initiate replication at oriC to converge on the terminus.Twin domains of supercoiling predict that the terminus couldexperience a loss of negative supercoiling or might even becomepositively supercoiled during fork convergence (25, 73). Inaddition, dimeric chromosomes that arise from reciprocal recombinationmust be resolved to monomers by site-specific recombinationat dif prior to cell division (45, 52).

To study supercoiling at dif, transposons with associated ressites were isolated near dif. The resolvase system uses movementof plectonemic negative supercoils to form a recombination synapse—athree-node superhelix that pairs two 120-bp resolution sites(res sites) (65). Resolution reactions were studied at threedifferent locations, the his-cob interval, which is 468 kb fromdif (37), an interval within 3 kb of the dif site, and a thirdinterval in the opposite replichore 600 kb from dif near moeA(Table 1 and Fig. 3A). Each interval is defined by two transposonswith a single res site (37). In strains with WT gyrase, resolutionwas efficient at the cob, dif, and moeA regions of the chromosome.Over 80% of the cells in exponentially growing populations generateda deletion after a 10-min induction of ${gamma}$ ${delta}$ resolvase expression(Fig. 3B, left panel). When resolution was tested in stationary-phasecells, essentially all chromosomes were recombinant (Fig. 3B,right panel). Thus, supercoil behavior in WT cells was similarat cob, dif, and moeA (Fig. 3A).

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FIG.3. A. Genetic and physical map of the S. enterica serovar Typhimurium chromosome. Intervals analyzed with Tn3 resolution assays are shown for seven map positions on the standard 100-CS Salmonella map. Replichore I goes from oriC clockwise, while replichore II goes counterclockwise. The map coordinates for intervals defined by transposon insertions are indicated by black triangles, and inserted Lac modules are indicated by diamonds. B. The data for resolution efficiencies shown in Table 2 for three transposon-defined intervals at CS 43, CS 33, and CS 19 are shown as striped bars for WT gyrase and black bars for GyrB652. Resolution efficiencies for deletion of Lac intervals are indicated by white bars for WT gyrase and gray bars for GyrB652. C. Relative to WT gyrase, the reduction in recombination efficiency for GyrB652 derivatives is shown for all chromosomal positions.

However, gyrB652 derivatives had a markedly different profile(Fig. 3B). In exponential-phase cells, resolution near dif wasreduced 25-fold compared to WT cells. This reduction in resolutionefficiency was much greater than that observed for intervalsof comparable size at the cob operon (ninefold), which is about450 kb clockwise on the standard map, or the interval near moeA,which is 600 kb counterclockwise of dif (fivefold) (Fig. 3B;Table 2). In stationary phase, GyrB652 strains at the cob andmoeA regions had only 1.5- and 2-fold reductions relative toWT, but a 10-fold reduction in resolution was still seen neardif (Fig. 3B, right panel).

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TABLE 2. Effect of gyrB652 mutation on resolution in different chromosome regions^a

Resolution assays at three chromosomal locations showed differencesin supercoiling caused by the GyrB652 mutant. These assays monitorthe loss of the MudJr2 element, which includes a lacZ gene anda Kan resistance gene. However, each interval is slightly differentin size and there might be supercoil influences associated withthe bacterial genes altered by transposon insertions or causedby the resulting deletion. To eliminate these complications,a copy of the complete E. coli lac operon, flanked by directlyrepeated res sites, was introduced into four intergenic locationsof the Salmonella chromosome (Fig. 4; see Materials and Methods).Resolution of the 9-kb Lac-Gen module was tested at four positionsin the replichore II segment of the Salmonella chromosome. Thesepositions were near centisome (CS) 33 (which is within 3 kbof dif), CS 58, CS 71, and CS 85 (which is near oriC) (Fig.3A). By measuring deletion of the same interval at multiplelocations, we eliminated minor variations in comparisons thatare due to different segment lengths and no Salmonella genewas disrupted. Data from these four intervals confirmed andextended our conclusions from the three transposon-defined intervalsat CS 43, CS 33, and CS 19. First, resolution of all intervalswas 80% or greater in WT cells when cells were tested in logphase, and it was essentially 100% in stationary cultures (Fig.3B). As shown by others working with both E. coli and Salmonella,supercoil structure in WT cells is similar in all regions ofthe genome (48, 51). However, in the GyrB652 mutant, exponentialcells exhibited a gradient of supercoil disruption (Fig. 3B).The region least affected was near oriC (22% resolution efficiency),and the zone with the most severe loss of supercoil structurewas near dif (4% resolution; Fig. 3B, left panel). In stationary-phasecultures, resolution near oriC was unaltered by GyrB652 (97%),whereas the resolution for the dif site remained low (22%; Fig.3B, right panel). When these results are expressed as an n-foldreduction in recombination relative to strains with WT gyrase,the interval near dif showed 20- to 25-fold decreases for resolutionin log phase, whereas the oriC region showed a modest fourfolddecrease (Fig. 3C).

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FIG. 4. Introduction of a Lac-Gent module by FLP recombination. A FRT site, which includes a 34-bp FRT core sequence, was introduced into the S. enterica serovar Typhimurium genome using the bacteriophage ${lambda}$ red recombination system (14, 74). Strains containing a single FRT site and the plasmid pCP20 (not shown), which contains a thermoinducible FLP recombinase, were induced for FLP expression and immediately transformed by electroporation with the 12-kb plasmid pPM1 (shown at bottom left). FLP recombination releases a 9.2-kb FRT-generated circle (bottom right) which can recombine into the chromosomal FRT site to generate a gentamicin-resistant strain with a lac operon flanked by res and FRT sites (top).

DISCUSSION

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Discussion

Gyrase formed with the GyrB652 subunit is not a TS enzyme invitro or in vivo (Fig. 1 and 2). Rather, GyrB652 gyrase hasa poor k_cat relative to the WT gyrase at 30°C, 37°C,and 42°C (Fig. 1 and 2). The gyrB652 mutation resides ina region of the protein that interacts with the GyrA subunit(8, 42, 56). Thus, the mutation may make gyrase less efficientby impeding interactions between the A and B subunits. The two-to fourfold effect of the gyrB652 mutation on the enoxacin-dependentshift to a cleavable complex (Fig. 2) matches the specific activitythat is three- to fivefold lower than for WT GyrB in supercoilingassays conducted at 30°C. Defining the precise loss in catalyticefficiency is important but difficult. Single-molecule assays(15) for negative supercoiling are not yet available (N. R.Cozzarelli, personal communication). Nonetheless, contacts betweenprotein subunits can clearly modulate enzyme efficiency. Forexample, protein-protein contacts between DNA-bound RecA protomersmay explain the differences in recombinational efficiency ofthe E. coli and Pseudomonas aeruginosa RecA proteins (4).

Since the TS response is not a failure of the enzyme to workat 42°C, why do cells stop growth? As a central enzyme inDNA metabolism, the catalytic rate of gyrase could be requiredto operate within specific limits to accommodate DNA compactionand cell division timing mechanisms. We suggest that three factorscould contribute to replication problems in gyrB hypomorphicmutants. First, even at permissive temperature (30°C) thenumber of supercoil domains per genome equivalent of DNA ina GyrB652 strain increases to over 1,000 from the 400 domainsfound in WT cells (17, 55, 66). Although our assay restrictsus to studies at 30°C because of the TS repressor controlon resolvase expression, others have shown that the number ofDNA genome equivalents per cell at the fastest growth ratesreaches a median between 2 and 4 (62). Under these conditions,unless gyrase expression increases, domains will exceed thenumber of cellular gyrase molecules (about 1,000/cell) by severalfold.Second, during chromosomal replication, oriC is physically separatedfrom the terminus during formation of sister nucleoids (5, 20,49, 68). Work from several labs shows that the physical separationof ori and the terminus is a highly reproducible part of a bacterialcell cycle (5, 20, 71). Third, the chromosomes in GyrB652 cellsshow a gradient of supercoil disruption in which the regionclosest to oriC is nearest to normal WT and the dif region isthe most disorganized (Fig. 3B to C). Because of a supercoil-sensingmechanism that drives the gyrB promoter (46, 53), gyrB's positionnear oriC may preclude it sensing a loss of supercoiling atdif.

Another factor may involve Topo IV (a tetramer of ParC and ParEproteins), which relaxes both positive and negative supercoilsin addition to decatenating the replicated sister chromosomes(13, 16, 22). Topo IV contributes to the supercoil balance invivo (75), and it becomes concentrated near the terminus ofreplication (21), possibly occupying a high-affinity site neardif (38). Topo IV relaxation activity near dif would exacerbatethe loss of gyrase-dependent supercoiling in a GyrB652 mutant.

Dichotomous chaos and the terminus. In addition to discovering the complex GyrB652 phenotype, Gariet al. found that cell growth is required for gyrase mutantsto trigger an SOS response (26). A genetic selection for cellsthat survive recA- or recBCD-gyrA208 synthetic lethality revealedTS mutants in tRNA aminoacyl synthetases. When protein synthesiswas blocked soon after temperature up-shift, cells were protectedfrom synthetic lethality and SOS induction. Garil et al. (26)proposed that chromosome breaks were caused by unbalanced growthand suggested a link to thymine-less death that occurs in bothprokaryotes and eukaryotes (3). We propose that the factorslisted above contribute to unbalanced growth during dichotomousgrowth, which is the poly-fork DNA replication pattern thataccompanies growth of E. coli and Salmonella in rich mediumat a high temperature (Fig. 5A to C). As growth rate increases,cells reinitiate replication before the initial round is complete.Initiation at oriC (Fig. 5A) leads to bidirectional synthesiswith two replisomes (labeled 1 and 2 in Fig. 5A) proceedingin opposite directions (7). When replication proceeds to a specifieddistance from the origin, initiation occurs again at each replicatedoriC. This adds four new replication forks (3 to 6 in Fig. 5B),and at cell division in WT cells, two daughters inherit a completechromosome and partially replicated chromosomes (data not shown).The products of two rounds of initiation are four chromosomeswith strands of the original template, strands from the firstinitiation, and strands from the second initiation (Fig. 5C).

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FIG. 5. Model for topological chaos at dif and SOS and Rec-less degradation in a GyrB652 mutant. A. In strains with WT gyrase, initiation at oriC (0) leads to semiconservative (red strands) and bidirectional replication using two forks (labeled 1and 2). B. At 42°C in rich medium, rapid cell growth allows a second round of replication initiates at each oriC leading to four more forks (labeled 3, 4, 5, and 6) and new blue strands of DNA. C. As replication forks 1and 2 complete synthesis at the terminus, cell division proceeds with daughter cells inheriting two complete and two partially replicated chromosomes (not shown). When the second round is complete, four genome equivalents are produced with two black/blue strands and two red/blue strands. D. Strains with a GyrB652 gyrase have well organized origin regions and poorly organized termini. Collapse of replication fork 1 stalls replichore II while fork movement through replichore I continues unabated. E. Rapid cell growth triggers reinitiation at both oriCs. If forks 3 and 5 overtake stalled fork 1, double-strand ends expose red/blue strands to RecBCD degradation. The residual elements of forks 3 and 5 (black/blue strands) can restart one fork while forks 4 and 6 proceed to the terminus. In a RecA mutant, rescue of DNA ends by strand invasion is impaired, which leads to degradation of the partial chromosomal replicas with red/blue strands. F. The outcome is two genome equivalents of DNA having both an original template (black) and one blue strand derived from the second initiation.

All barriers to the DnaB helicase must be eliminated to allowunimpeded fork movement at the normal rate of >800 bp/s.We hypothesize that DNA replication in cells carrying the sluggishhypomorphic gyrase bogs down due to poor supercoil condensationof DNA near the terminus. If a fork collapses while cell growthcontinues (10, 47), there is a critical time for restartingbefore new forks overtake the stalled fork (Fig. 5). When asecond fork runs into a stalled fork, it exposes a double-strandedend (see Bidnenko et al. [6]). This won't happen nearly as muchif DNA initiation is blocked or delayed by slow protein synthesis,and chromosomal termini can gain time to condense and resolveDNA at low fork density. The condition of a GyrB652 mutant mimicsthe killing of cells with quinolones, which also requires growthand protein synthesis for achieving a lethal effect (39).

The terminal chaos hypothesis is consistent with several recentobservations. In an E. coli gyrase hypomorph, Jeong et al. founda large disruption in gene expression near dif, and their chromosomeimmunoprecipitation assays indicated gyrase depletion near theterminus (41). Moreover, several genetic conditions that increaseoriC initiation also trigger fork failure. One example is theoverproduction of DnaA, which leads to overinitiation, forkcollapse (29), and breakage of chromosomal DNA with loss ofcell viability (61). Terminal chaos can explain the sicknessof SeqA mutants, which must be grown in minimal medium at alow temperature because they lack the mechanism by which SeqAinhibits premature reinitiation by blocking access of DnaA proteinto oriC (9, 63). In addition to gyrase, SeqA, and DnaA, phenotypesof growth rate toxicity are found with mutants of priA (58,59), mukB (1), diaA (40), and parC and parE (2). If resolutionnear dif is specifically impaired in several of these mutants,then defining a comprehensive set of genes that change domainstructure at high growth rates would be an important step towardcompleting our understanding of DNA fork termination and chromosomesegregation.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantGM-33143-20 and MCB 0110675 from the National Science Foundation.

We thank N. Bossi, N. Figueroa, and M. Schmid for the gift ofmutant strains.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294. Phone: (205) 934-3299. Fax: (205) 975-5955. E-mail: nphiggin{at}uab.edu.

REFERENCES

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