Genetic studies on the role of type IA DNA topoisomerases in DNA metabolism and genome maintenance in Escherichia coli

Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes.DNA supercoiling is important for all cellular processes that require strand separation and is regulated by the opposing enzymatic effects of DNA topoisomerases. Gyrase uses ATP to introduce negative supercoils while topoisomerase I (topA) and topoisomerase IV relax negative supercoils. Cells lacking topoisomerase I are only viable if they have compensatory mutations in gyrase genes that reduce the negative supercoiling level of the chromosome to allow bacterial growth. One such mutation leads to the production of a thermosensitive gyrase (gyrB(Ts)). Gyrase driven supercoiling during transcription in the absence of topoisomerase I causes the accumulation of hypernegatively supercoiled plasmid DNAs due to the formation of R-loops. Overproducing RNase HI (rnhA), an enzyme that degrades the RNA moiety of R-loops, prevents the accumulation of hypernegative supercoils. In the absence of RNase HI alone, R-loops are equally formed and can be used to prime DNA replication independently of oriC/DnaA, a phenomenon known as constitutive stable DNA replication (cSDR). To better understand the link between R-loop formation and hypernegative supercoiling, we constructed a conditional topA rnhA gyrB(Ts) mutant with RNase HI being conditionally expressed from a plasmid borne gene. We found that the DNA of topA rnhA gyrB(Ts) cells was extensively relaxed instead of being hypernegatively supercoiled following the depletion of RNase HI. Relaxation was found to be unrelated to the activity of topoisomerase IV. Cells of topA rnhA gyrB(Ts) formed long filaments full of DNA, consistent with segregation defect. Overproducing topoisomerase III (topB), an enzyme that can perform decatenation, corrected the segregation problems without restoring supercoiling. We found that extracts of topA rnhA gyrB(Ts) cells inhibited gyrase supercoiling activity of wild type cells extracts in vitro, suggesting that the depletion of RNase HI triggered a cell response that inhibited the supercoiling activity of gyrase. Gyrase supercoiling assays in vivo as well as in crude cell extracts revealed that the ATP dependent supercoiling reaction of gyrase was inhibited while the ATP independent relaxation reaction was unaffected. Genetic suppressors of a triple topA rnhA gyrB(Ts) strain that restored supercoiling and corrected the chromosome segregation defects mostly mapped to genes that affected DNA replication, R-loop metabolism and fimbriae formation. The second part of this project aimed at understanding the roles of type IA DNA topoisomerases (topoisomerase I and topoisomerase III) in chromosome segregation and genome maintenance in E. coli. To investigate the role of type IA DNA topoisomerases in chromosome segregation we employed genetic approaches combined with flow cytometry, Western blot analysis and microscopy (for the examination of cell morphology). We found that the Par- phenotypes (formation of large unsegregated nucleoid in midcell) and chromosome segregation defects of a gyrB(Ts) mutant at the nonpermissive temperature were corrected by deleting topA only in the presence of topB. Moreover, overproducing topoisomerase III was shown to correct the Par- phenotype without correcting the growth defect, but overproducing topoisomerase IV, the major cellular decatenase, failed to correct the defects. Our results suggest that type IA topoisomerases play a role in chromosome segregation when gyrase is inefficient. To investigate the role of type IA DNA topoisomerases in genome maintenance, in the third part of the project, we employed genetic approaches combined with suppressor screens, spot assays and microscopy. We found that cells lacking topoisomerase I suffered from supercoiling-dependent growth defects and chromosome segregation defects that could be corrected by deleting recQ, recA or overproducing topoisomerase III and by an oriC15::aph suppressor mutation isolated in the first part of the project. Cells lacking both type 1A topoisomerases formed very long filaments packed with diffuse and unsegregated DNA. Such phenotypes could be partially corrected by overproducing RNase HI or deleting recA, or by suppressor mutations isolated in the first part of the project, that affected cSDR (dnaT18::aph and rne59::aph). Thus, in E. coli, type IA DNA topoisomerases play a role in genome maintenance by inhibiting inappropriate replication from oriC and R-loops and by preventing RecA-dependent chromosome segregation defect through their action with RecQ. The work reported here reveals that inappropriate and unregulated replication is a major source of genome instability. Preventing such replication will ensures proper chromosome segregation leading to a stable genome. RNase HI and type IA DNA topoisomerases play a leading role in preventing unregulated replication. RNase HI achieves this role by modulating ATP dependent gyrase activity and by preventing replication from R-loops (cSDR). Type IA DNA topoisomerases ensure the maintenance of a stable genome by preventing inappropriate replication from oriC and R-loops and by acting with RecQ to prevent RecA dependent-chromosome segregation defects

Colistin Induces S. aureus Susceptibility to Bacitracin

Bacitracin has been used in topical preparations with polymyxin B for bacterial infections. Colistin belongs to the polymyxin group of antibiotics and is effective against most Gram-negative bacilli. This study investigated whether colistin could affect the susceptibility of S. aureus to bacitracin. S. aureus isolates were first incubated with colistin and the susceptibility of S. aureus to bacitracin was increased. The effect of the combination of colistin and bacitracin on S. aureus was then confirmed by the checkerboard assay and the time-kill kinetics. The Triton X-100-induced autolysis was significantly increased after S. aureus was exposed to colistin. Exposure to colistin also led to a less positive charge on the cell surface and a significant leakage of Na+, Mg2, K+, Ca2+, Mn2+, Cu2+, and Zn2+. Finally, disruptions on the cell surface and an irregular morphology were observed when the bacteria were exposed to colistin and bacitracin. Bacitracin had a stronger antibacterial activity against S. aureus in the presence of colistin. This could be due to the fact that colistin damaged the bacterial membrane. This study suggests that combination of colistin with bacitracin has a potential for treating clinical S. aureus infections

Topo III overproduction and <i>recQ</i> deletion are epistatic to <i>recA</i> deletion in correcting the growth defect of the <i>topA gyrB</i>(Ts) strain.

<p>Spot tests were performed at the indicated temperatures. The LB plates were incubated for the indicated times. The strains used are all derivatives of RFM475 (<i>gyrB</i>(Ts) <i>ΔtopA</i>). They are: a) CT150 (RFM475 <i>ΔrecQ</i>), SB265 (RFM475 <i>ΔrecA</i>) and VU492 (<i>ΔrecQ ΔrecA</i>); b) VU118 (RFM475/pPH1243), SB265 (RFM475 <i>ΔrecA</i>) and VU479 (SB265/pPH1243); c) VU118 (RFM475/pPH1243), CT150 (RFM475 <i>ΔrecQ</i>) and VU464 (CT150/pPH1243); d) RFM475, SB362 (RFM475 <i>lexA3</i>) and SB265 (RFM475 <i>ΔrecA</i>); e) SB362 (RFM475 <i>lexA3</i>), CT150 (RFM475 <i>ΔrecQ</i>) and VU501 (RFM475 <i>ΔrecQ lexA3</i>); f) RFM475 and SB262 (RFM475 <i>recB</i>::Tn<i>10</i>). Cells carrying pPH1243 were grown in the presence of IPTG to overproduce topo III.</p

Effects of RNase HI overproduction and <i>recA</i> and <i>recQ</i> deletions on cells lacking type 1A topos.

<p>(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at 30°C as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#s4" target="_blank">Materials and Methods</a>. Size bars are 5 µm. (b) Spot tests at 30°C. The LB plate was incubated for 24 h. The strains used are all derivative of RFM475 (<i>gyrB</i>(Ts) <i>ΔtopA</i>). They are: VU306 (RFM475 <i>ΔtopB</i>/pSK760), VU333 (RFM475 <i>ΔtopB</i>/pSK762c), VU363 (RFM475 <i>ΔtopB ΔrecQ</i>/pSK760), VU365 (RFM475 <i>ΔtopB ΔrecQ</i>/pSK762c), VU375 (RFM475 <i>ΔtopB ΔrecA</i>/pSK760) and VU379 (RFM475 <i>ΔtopB ΔrecA</i>/pSK762c). pSK760 carries the <i>rnhA</i> gene for RNase HI overproduction, whereas pSK762c carries a mutated and inactive <i>rnhA</i> gene.</p

Replication initiation asynchrony and reduced DNA/mass ratio conferred by the <i>oriC15</i>::<i>aph</i> suppressor mutation.

<p>(a) Schematic representation of the minimal <i>oriC</i> region (245 bp) with its regulatory elements. DUE is the DNA unwinding element with its AT-cluster and 13-mer repeats L, M, and R (orange). DnaA binding sites: R1, R2 and R4 are high affinity sites (blue) whereas R3, R5, I1-3 and τ1-2 are low affinity sites (yellow). I1-3 and τ1-2 preferentially bind DnaA-ATP. IHF and FIS binding sites are also shown. For more details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Leonard1" target="_blank">[58]</a>. <i>aph</i> indicates the insertion site of the <i>kan^r</i> cassette in our <i>oriC15::aph</i> insertion mutant (position 142 in the 245 bp <i>oriC</i> region). The <i>oriC231</i> allele of Stepankiw <i>et al</i>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Stepankiw1" target="_blank">[57]</a> spanning the left portion of <i>oriC</i> up to the arrow is shown for comparison (position 163 in the 245 bp <i>oriC</i> region). (b) Rifampicin run-out experiments for flow cytometry analysis were performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#s4" target="_blank">Materials and Methods</a>. Cells were grown in M9 minimal medium. (c) DNA/mass ratios were calculated as described in Material and Methods from three independent flow cytometry experiments. The strains used were: RFM443 (wild-type), RFM445 (<i>gyrB</i>(Ts)), RFM475 (<i>gyrB</i>(Ts) <i>ΔtopA</i>) and VU155 (RFM475 <i>oriC15::aph</i>). RFM475 has a significantly higher (*) DNA/mass ratio compared to RFM445 (p = 0.0199), RFM443 (p = 0.0274) and RFM475 <i>oriC</i> (p = 0.0292) Moreover, there is no statistical differences between the DNA/mass ratio of RFM475 <i>oriC</i> compared to RFM445 (p = 0.6587) and RFM443 (p = 0.8798).</p

Roles of Type 1A Topoisomerases in Genome Maintenance in <i>Escherichia coli</i>

<div><p>In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the <i>E. coli</i> topo I (<i>topA</i>) and III (<i>topB</i>) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that <i>topA</i> mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting <i>recA</i> or <i>recQ</i> but not by deleting <i>recJ</i> or <i>recO</i>, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-<i>recQ</i> deletion and topo IIII overproduction are epistatic to <i>recA</i> deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an <i>oriC</i>::<i>aph</i> suppressor mutation which is <i>oriC</i>-competent in <i>topA</i> null but not in isogenic <i>topA⁺</i> cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double <i>topA rnhA</i> mutants affecting constitutive stable DNA replication, <i>dnaT</i>::<i>aph</i> and <i>rne</i>::<i>aph</i>, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from <i>oriC</i> (topo I alone) and R-loops and (ii) by acting with RecQ.</p></div

Growth and chromosome segregation defects in the <i>gyrB</i>(Ts) <i>ΔtopA</i> strain.

<p>(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#s4" target="_blank">Materials and Methods</a>. Size bars are 5 µm. Additional images are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s002" target="_blank">S2</a>. (b) Spot tests were performed at the indicated temperatures. The LB plates were incubated for the indicated times. The strains used are RFM475 (<i>gyrB</i>(Ts) <i>ΔtopA</i>), RFM445 (<i>gyrB</i>(Ts)) and VU287 (RFM475/pSK760). pSK760 carries the <i>rnhA</i> gene for RNase HI overproduction.</p

Topo III overproduction, and <i>recA</i> and <i>recQ</i> deletions complement the growth and chromosome segregation defects in the <i>gyrB</i>(Ts) <i>ΔtopA</i> strain.

<p>(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#s4" target="_blank">Materials and Methods</a>. Size bars are 5 µm. Additional images are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s002" target="_blank">S2</a>. (b) Spot tests were performed at the indicated temperatures. The LB plates were incubated for the indicated times. The strains used are all derivatives of RFM475 (<i>ΔtopA gyrB(</i>Ts)) except SB264 which is a derivative of RFM445 (<i>gyrB</i>(Ts)). They are: CT150 (RFM475 <i>ΔrecQ</i>), VU118 (RFM475/pPH1243), SB265 (RFM475 <i>ΔrecA</i>), VU454 (RFM475 <i>ΔrecO</i>) and SB264 (RFM445 <i>ΔrecA</i>). Cells carrying pPH1243 were grown in the presence of IPTG to overproduce topo III.</p

The <i>dnaT18</i>::<i>aph</i> and <i>rne59</i>::<i>aph</i> suppressor mutations inhibit cSDR in an <i>rnhA</i> strain.

<p>(a) Model for constitutive stable DNA replication (cSDR) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Kogoma1" target="_blank">[55]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Sandler1" target="_blank">[61]</a>. R-loop forms during transcription when the nascent RNA hybridizes with the template DNA strand behind the moving RNA polymerase. Both transcription-induced negative supercoiling and RecA protein promote R-loop formation. DNA pol I synthesizes DNA from the 3′ end of the hybridized RNA for primosome (PriA-dependent) assembly. Eventually, the primosome allows the assembly of two replisomes for bidirectional replication. The proteins that are included in the present study are shown in red: topo I relaxes transcription-induced negative supercoiling; RecA promotes the hybridization of the template DNA strand with the nascent RNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Kasahara1" target="_blank">[86]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Zaitsev1" target="_blank">[87]</a>; RNase HI degrades the RNA of the R-loop; RNase E may inhibit R-loop formation by degrading the nascent RNA; DnaT may play a role in cSDR via the primosome. (b) A map of the <i>E. coli</i> chromosome showing the normal origin of replication (<i>oriC</i>), the putative cSDR origins of replication (<i>oriK</i>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Kogoma1" target="_blank">[55]</a>) and two of the ten <i>ter</i> sites, with <i>terC</i> believed to be a site where many convergent replication forks meet <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543-Duggin1" target="_blank">[88]</a>. (c) and (d). Spot tests. The LB plates were incubated for 24 h, at 30 or 42°C as indicated. The strains used were: MD48 (<i>dnaA46</i>(Ts)), JE35 (<i>rnhA dnaA46</i>(Ts)), VU204 (<i>dnaA46</i>(Ts), <i>dnaT</i>), VU200 (<i>rnhA dnaA46</i>(Ts) <i>dnaT</i>), JE36 (<i>rnhA dnaA46</i>(Ts) <i>rne</i>) and JE119 (<i>dnaA46</i>(Ts) <i>rne</i>). At 42°C, the few colonies of strain MD48 (at 10⁰ and 10⁻¹) were made of cells that have acquired compensatory mutations, as they grew robustly upon restreaking them at the same temperature.</p

Effects of mutations affecting DNA replication on the growth and chromosome segregation defects in the <i>gyrB</i>(Ts) <i>ΔtopA</i> strain.

<p>(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#s4" target="_blank">Materials and Methods</a>. Size bars are 5 µm. Additional images are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004543#pgen.1004543.s002" target="_blank">S2</a>. (b) Spot tests were performed at the indicated temperatures. The strains used are derivatives of RFM475 (<i>gyrB</i>(Ts) <i>ΔtopA</i>). They are: VU155 (RFM475 <i>oriC</i>), VU188 (RFM475 <i>dnaT</i>) and VU176 (RFM475 <i>holC2::aph</i>).</p