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. 2014 Aug 7;10(8):e1004543.
doi: 10.1371/journal.pgen.1004543. eCollection 2014 Aug.

Roles of type 1A topoisomerases in genome maintenance in Escherichia coli

Valentine Usongo  1 , Marc Drolet  1
Affiliations

Roles of type 1A topoisomerases in genome maintenance in Escherichia coli

Valentine Usongo et al. PLoS Genet. .

Abstract

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 E. coli topo I (topA) and III (topB) 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 topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, 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-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ 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 topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Growth and chromosome segregation defects in the gyrB(Ts) ΔtopA strain.
(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures, as described in Materials and Methods. Size bars are 5 µm. Additional images are shown in Figure S1 and S2. (b) Spot tests were performed at the indicated temperatures. The LB plates were incubated for the indicated times. The strains used are RFM475 (gyrB(Ts) ΔtopA), RFM445 (gyrB(Ts)) and VU287 (RFM475/pSK760). pSK760 carries the rnhA gene for RNase HI overproduction.
Figure 2
Figure 2. Topo III overproduction, and recA and recQ deletions complement the growth and chromosome segregation defects in the gyrB(Ts) ΔtopA strain.
(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures as described in Materials and Methods. Size bars are 5 µm. Additional images are shown in Figure S1 and S2. (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 (ΔtopA gyrB(Ts)) except SB264 which is a derivative of RFM445 (gyrB(Ts)). They are: CT150 (RFM475 ΔrecQ), VU118 (RFM475/pPH1243), SB265 (RFM475 ΔrecA), VU454 (RFM475 ΔrecO) and SB264 (RFM445 ΔrecA). Cells carrying pPH1243 were grown in the presence of IPTG to overproduce topo III.
Figure 3
Figure 3. Topo III overproduction and recQ deletion are epistatic to recA deletion in correcting the growth defect of the topA gyrB(Ts) strain.
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 (gyrB(Ts) ΔtopA). They are: a) CT150 (RFM475 ΔrecQ), SB265 (RFM475 ΔrecA) and VU492 (ΔrecQ ΔrecA); b) VU118 (RFM475/pPH1243), SB265 (RFM475 ΔrecA) and VU479 (SB265/pPH1243); c) VU118 (RFM475/pPH1243), CT150 (RFM475 ΔrecQ) and VU464 (CT150/pPH1243); d) RFM475, SB362 (RFM475 lexA3) and SB265 (RFM475 ΔrecA); e) SB362 (RFM475 lexA3), CT150 (RFM475 ΔrecQ) and VU501 (RFM475 ΔrecQ lexA3); f) RFM475 and SB262 (RFM475 recB::Tn10). Cells carrying pPH1243 were grown in the presence of IPTG to overproduce topo III.
Figure 4
Figure 4. Replication initiation asynchrony and reduced DNA/mass ratio conferred by the oriC15::aph suppressor mutation.
(a) Schematic representation of the minimal oriC 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 . aph indicates the insertion site of the kanr cassette in our oriC15::aph insertion mutant (position 142 in the 245 bp oriC region). The oriC231 allele of Stepankiw et al. spanning the left portion of oriC up to the arrow is shown for comparison (position 163 in the 245 bp oriC region). (b) Rifampicin run-out experiments for flow cytometry analysis were performed as described in Materials and Methods. 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 (gyrB(Ts)), RFM475 (gyrB(Ts) ΔtopA) and VU155 (RFM475 oriC15::aph). RFM475 has a significantly higher (*) DNA/mass ratio compared to RFM445 (p = 0.0199), RFM443 (p = 0.0274) and RFM475 oriC (p = 0.0292) Moreover, there is no statistical differences between the DNA/mass ratio of RFM475 oriC compared to RFM445 (p = 0.6587) and RFM443 (p = 0.8798).
Figure 5
Figure 5. Effects of mutations affecting DNA replication on the growth and chromosome segregation defects in the gyrB(Ts) ΔtopA strain.
(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at the indicated temperatures as described in Materials and Methods. Size bars are 5 µm. Additional images are shown in Figure S1 and S2. (b) Spot tests were performed at the indicated temperatures. The strains used are derivatives of RFM475 (gyrB(Ts) ΔtopA). They are: VU155 (RFM475 oriC), VU188 (RFM475 dnaT) and VU176 (RFM475 holC2::aph).
Figure 6
Figure 6. Effects of RNase HI overproduction and recA and recQ deletions on cells lacking type 1A topos.
(a) Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at 30°C as described in Materials and Methods. 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 (gyrB(Ts) ΔtopA). They are: VU306 (RFM475 ΔtopB/pSK760), VU333 (RFM475 ΔtopB/pSK762c), VU363 (RFM475 ΔtopB ΔrecQ/pSK760), VU365 (RFM475 ΔtopB ΔrecQ/pSK762c), VU375 (RFM475 ΔtopB ΔrecA/pSK760) and VU379 (RFM475 ΔtopB ΔrecA/pSK762c). pSK760 carries the rnhA gene for RNase HI overproduction, whereas pSK762c carries a mutated and inactive rnhA gene.
Figure 7
Figure 7. The dnaT18::aph and rne59::aph suppressor mutations inhibit cSDR in an rnhA strain.
(a) Model for constitutive stable DNA replication (cSDR) , . 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 , ; 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 E. coli chromosome showing the normal origin of replication (oriC), the putative cSDR origins of replication (oriK, [55]) and two of the ten ter sites, with terC believed to be a site where many convergent replication forks meet . (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 (dnaA46(Ts)), JE35 (rnhA dnaA46(Ts)), VU204 (dnaA46(Ts), dnaT), VU200 (rnhA dnaA46(Ts) dnaT), JE36 (rnhA dnaA46(Ts) rne) and JE119 (dnaA46(Ts) rne). 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.
Figure 8
Figure 8. Effects of dnaT18::aph, rne59::aph and holC2::aph suppressor mutations on cells lacking type 1A topos.
Representative superimposed images of DIC and fluorescence pictures of DAPI-stained cells grown at 30°C as described in Materials and Methods. Size bars are 5 µm (a). Spot tests at 30°C (b), 24°C (c) and 37°C (d). The LB plates were incubated for the indicated times. The strains used are all derivative RFM445 ΔtopB (strain VU409: gyrB(Ts), ΔtopB). They are: VU421 (VU409 topA20::Tn10), VU422 (VU409 topA20::Tn10/pSK760), VU425 (VU409 topA20::Tn10/pSK762c), VU441 (VU409 dnaT topA20::Tn10), VU469 (VU409 holC topA20::Tn10), VU473 (VU409 rne topA20::Tn10). pSK760 carries the rnhA gene for RNase HI overproduction, whereas pSK762c carries a mutated and inactive rnhA gene.
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This work was supported by a Discovery Grant from NSERC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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