37 research outputs found
Genetic studies on the role of type IA DNA topoisomerases in DNA metabolism and genome maintenance in Escherichia coli
Le surenroulement de lâADN est important pour tous les processus cellulaires qui requiĂšrent la sĂ©paration des brins de lâADN. Il est rĂ©gulĂ© par lâactivitĂ© enzymatique des topoisomĂ©rases. La gyrase (gyrA et gyrB) utilise lâATP pour introduire des supertours nĂ©gatifs dans lâADN, alors que la topoisomĂ©rase I (topA) et la topoisomĂ©rase IV (parC et parE) les Ă©liminent. Les cellules dĂ©ficientes pour la topoisomĂ©rase I sont viables si elles ont des mutations compensatoires dans un des gĂšnes codant pour une sous-unitĂ© de la gyrase. Ces mutations rĂ©duisent le niveau de surenroulement nĂ©gatif du chromosome et permettent la croissance bactĂ©rienne. Une de ces mutations engendre la production d'une gyrase thermosensible. LâactivitĂ© de surenroulement de la gyrase en absence de la topoisomĂ©rase I cause lâaccumulation dâADN hyper-surenroulĂ© nĂ©gativement Ă cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dĂ©grade lâARN des R-loops, permet de prĂ©venir lâaccumulation dâun excĂšs de surenroulement nĂ©gatif. En absence de RNase HI, des R-loops sont aussi formĂ©s et peuvent ĂȘtre utilisĂ©s pour dĂ©clencher la rĂ©plication de lâADN indĂ©pendamment du systĂšme normal oriC/DnaA, un phĂ©nomĂšne connu sous le nom de « constitutive stable DNA replication » (cSDR).
Pour mieux comprendre le lien entre la formation de R-loops et lâexcĂšs de surenroulement nĂ©gatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec lâexpression inductible de la RNase HI Ă partir dâun plasmide. Nous avons trouvĂ© que lâADN des cellules de ce mutant Ă©tait excessivement relĂąchĂ© au lieu d'ĂȘtre hypersurenroulĂ© nĂ©gativement en conditions de pĂ©nurie de RNase HI. La relaxation de lâADN a Ă©tĂ© montrĂ©e comme Ă©tant indĂ©pendante de l'activitĂ© de la topoisomĂ©rase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de trĂšs longs filaments remplis dâADN, montrant ainsi un dĂ©faut de sĂ©grĂ©gation des chromosomes. La surproduction de la topoisomĂ©rase III (topB), une enzyme qui peut effectuer la dĂ©catĂ©nation de lâADN, a corrigĂ© les problĂšmes de sĂ©grĂ©gation sans toutefois restaurer le niveau de surenroulement de lâADN. Nous avons constatĂ© que des extraits protĂ©iques du mutant topA rnhA gyrB(Ts) pouvaient inhiber lâactivitĂ© de surenroulement nĂ©gatif de la gyrase dans des extraits dâune souche sauvage, suggĂ©rant ainsi que la pĂ©nurie de RNase HI avait dĂ©clenchĂ© une rĂ©ponse cellulaire dâinhibition de cette activitĂ© de la gyrase. De plus, des expĂ©riences in vivo et in vitro ont montrĂ© quâen absence de RNase HI, lâactivitĂ© ATP-dĂ©pendante de surenroulement nĂ©gatif de la gyrase Ă©tait inhibĂ©e, alors que lâactivitĂ© ATP-indĂ©pendante de cette enzyme demeurait intacte. Des suppresseurs extragĂ©niques du dĂ©faut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent Ă©galement les problĂšmes de surenroulement et de sĂ©grĂ©gation des chromosomes ont pour la plupart Ă©tĂ© cartographiĂ©s dans des gĂšnes impliquĂ©s dans la rĂ©plication de lâADN, le mĂ©tabolisme des R-loops, ou la formation de fimbriae.
La deuxiĂšme partie de ce projet avait pour but de comprendre les rĂŽles des topoisomĂ©rases de type IA (topoisomĂ©rase I et topoisomĂ©rase III) dans la sĂ©grĂ©gation et la stabilitĂ© du gĂ©nome de Escherichia coli. Pour Ă©tudier ces rĂŽles, nous avons utilisĂ© des approches de gĂ©nĂ©tique combinĂ©es avec la cytomĂ©trie en flux, lâanalyse de type Western blot et la microscopie. Nous avons constatĂ© que le phĂ©notype Par- et les dĂ©fauts de sĂ©grĂ©gation des chromosomes dâun mutant gyrB(Ts) avaient Ă©tĂ© corrigĂ©s en inactivant topA, mais uniquement en prĂ©sence du gĂšne topB. En outre, nous avons dĂ©montrĂ© que la surproduction de la topoisomĂ©rase III pouvait corriger le phĂ©notype Par- du mutant gyrB(Ts) sans toutefois corriger les dĂ©fauts de croissance de ce dernier. La surproduction de topoisomĂ©rase IV, enzyme responsable de la dĂ©catĂ©nation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomĂ©rase III. Nos rĂ©sultats suggĂšrent que les topoisomĂ©rases de type IA jouent un rĂŽle important dans la sĂ©grĂ©gation des chromosomes lorsque la gyrase est inefficace.
Pour Ă©tudier le rĂŽle des topoisomĂ©rases de type IA dans la stabilitĂ© du gĂ©nome, la troisiĂšme partie du projet, nous avons utilisĂ© des approches gĂ©nĂ©tiques combinĂ©es avec des tests de « spot » et la microscopie. Nous avons constatĂ© que les cellules dĂ©ficientes en topoisomĂ©rase I avaient des dĂ©fauts de sĂ©grĂ©gation de chromosomes et de croissance liĂ©s Ă un excĂšs de surenroulement nĂ©gatif, et que ces dĂ©fauts pouvaient ĂȘtre corrigĂ©s en inactivant recQ, recA ou par la surproduction de la topoisomĂ©rase III. Le suppresseur extragĂ©nique oriC15::aph isolĂ© dans la premiĂšre partie du projet pouvait Ă©galement corriger ces problĂšmes. Les cellules dĂ©ficientes en topoisomĂ©rases de type IA formaient des trĂšs longs filaments remplis dâADN dâapparence diffuse et rĂ©parti inĂ©galement dans la cellule. Ces phĂ©notypes pouvaient ĂȘtre partiellement corrigĂ©s par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolĂ©s dans la premiĂšre partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomĂ©rases de type IA jouent un rĂŽle dans la stabilitĂ© du gĂ©nome en inhibant la rĂ©plication inappropriĂ©e Ă partir de oriC et de R-loops, et en empĂȘchant les dĂ©fauts de sĂ©grĂ©gation liĂ©s Ă la recombinaison RecA-dĂ©pendante, par leur action avec RecQ.
Les travaux rapportĂ©s ici rĂ©vĂšlent que la rĂ©plication inappropriĂ©e et dĂ©rĂ©gulĂ©e est une source majeure de lâinstabilitĂ© gĂ©nomique. EmpĂȘcher la rĂ©plication inappropriĂ©e permet la sĂ©grĂ©gation des chromosomes et le maintien dâun gĂ©nome stable. La RNase HI et les topoisomĂ©rases de type IA jouent un rĂŽle majeur dans la prĂ©vention de la rĂ©plication inappropriĂ©e. La RNase HI rĂ©alise cette tĂąche en modulant lâactivitĂ© de surenroulement ATP-dependante de la gyrase, et en empĂȘchant la rĂ©plication Ă partir des R-loops. Les topoisomĂ©rases de type IA assurent le maintien de la stabilitĂ© du gĂ©nome en empĂȘchant la rĂ©plication inappropriĂ©e Ă partir de oriC et des R-loops et en agissant avec RecQ pour rĂ©soudre des intermĂ©diaires de recombinaison RecA-dĂ©pendants afin de permettre la sĂ©grĂ©gation 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>ÎtopA</i>). They are: a) CT150 (RFM475 <i>ÎrecQ</i>), SB265 (RFM475 <i>ÎrecA</i>) and VU492 (<i>ÎrecQ ÎrecA</i>); b) VU118 (RFM475/pPH1243), SB265 (RFM475 <i>ÎrecA</i>) and VU479 (SB265/pPH1243); c) VU118 (RFM475/pPH1243), CT150 (RFM475 <i>ÎrecQ</i>) and VU464 (CT150/pPH1243); d) RFM475, SB362 (RFM475 <i>lexA3</i>) and SB265 (RFM475 <i>ÎrecA</i>); e) SB362 (RFM475 <i>lexA3</i>), CT150 (RFM475 <i>ÎrecQ</i>) and VU501 (RFM475 <i>ÎrecQ 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 ”m. (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>ÎtopA</i>). They are: VU306 (RFM475 <i>ÎtopB</i>/pSK760), VU333 (RFM475 <i>ÎtopB</i>/pSK762c), VU363 (RFM475 <i>ÎtopB ÎrecQ</i>/pSK760), VU365 (RFM475 <i>ÎtopB ÎrecQ</i>/pSK762c), VU375 (RFM475 <i>ÎtopB ÎrecA</i>/pSK760) and VU379 (RFM475 <i>ÎtopB ÎrecA</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 Ï1-2 are low affinity sites (yellow). I1-3 and Ï1-2 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<sup>r</sup></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>ÎtopA</i>) and VU155 (RFM475 <i>oriC15::aph</i>). RFM475 has a significantly higher (*) DNA/mass ratio compared to RFM445 (pâ=â0.0199), RFM443 (pâ=â0.0274) and RFM475 <i>oriC</i> (pâ=â0.0292) Moreover, there is no statistical differences between the DNA/mass ratio of RFM475 <i>oriC</i> compared to RFM445 (pâ=â0.6587) and RFM443 (pâ=â0.8798).</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<sup>+</sup></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>ÎtopA</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 ”m. 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>ÎtopA</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>ÎtopA</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 ”m. 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>ÎtopA gyrB(</i>Ts)) except SB264 which is a derivative of RFM445 (<i>gyrB</i>(Ts)). They are: CT150 (RFM475 <i>ÎrecQ</i>), VU118 (RFM475/pPH1243), SB265 (RFM475 <i>ÎrecA</i>), VU454 (RFM475 <i>ÎrecO</i>) and SB264 (RFM445 <i>ÎrecA</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 3âČ 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 10<sup>0</sup> and 10<sup>â1</sup>) 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>ÎtopA</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 ”m. 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>ÎtopA</i>). They are: VU155 (RFM475 <i>oriC</i>), VU188 (RFM475 <i>dnaT</i>) and VU176 (RFM475 <i>holC2::aph</i>).</p