Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli

Abstract

Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.

Fig 1. Replication profiles of the wild-type strain RFM443 under various growth conditions.

Wild-type cells (RFM443) were grown at 37°C to log phase (top panel), stationary phase (middle panel) or log phase followed by a spectinomycin (spc) treatment (bottom panel), and genomic DNA was extracted for NGS as described in Materials and Methods. The absolute read counts (Log2) were plotted against chromosomal coordinates (W3110). The gray line is the loess regression curve (see Materials and Methods). The blue arrow points to the origin of replication (oriC) and the full and dashed black arrows respectively point to ydcM and lepA genes. The gap at position around 0.3 corresponds to the Δ(codB-lacI)3 deletion carried by the strains used in this work (see S1 Table).

Fig 2. Replication profiles of rnhA null strains.

rnhA::cam (MM84), rnhA::cam dnaT18::aph (JB04) and rnhA::cam IN(1.39–2.28) (AB004) cells were grown at 37°C to log phase and treated with spectinomycin (spc), and genomic DNA was extracted for NGS as described in Materials and Methods. The read counts (Log2) normalized against a wild-type (RFM443) spectinomycin treated control were plotted against chromosomal coordinates (W3110). The gray line is the loess regression curve (see Materials and Methods). The green arrows on the top of the profiles point to potential cSDR origins (oriKs) and the blue ones at the bottom of the profiles point to TerA and TerB polar replication termination sequences. The full and dashed black arrows in the top panel respectively point to ydcM and lepA genes. In the bottom panel, inv shows the chromosomal DNA inversion (1.39–2.28) in strain AB004.

Fig 3. Replication profiles of topA topB null strains.

topA20::Tn10 ΔtopB gyrB(Ts)/pSK762c (VU425), topA20::Tn10 ΔtopB gyrB(Ts)/pSK760 (VU422) cells were grown at 30°C to log phase and treated with spectinomycin (spc), and genomic DNA was extracted for NGS as described in Materials and Methods. The read counts (Log2) normalized against wild-type (RFM443) spectinomycin treated cells were plotted against chromosomal coordinates (W3110). The gray line is the loess regression curve (see Materials and Methods). The green arrows on the top of the profiles point to potential cSDR origins (oriKs) and the blue ones at the bottom of the profiles point to TerA and TerB polar replication termination sequences. The full and dashed black arrows respectively point to ydcM and lepA genes. amp shows the chromosomal DNA amplification (2.99–3.24) in strains VU422 and VU425.

Fig 4. ydcM/lepA ratio in E. coli rnhA, topA and topA topB null mutants.

Wild-type (RFM443) and rnhA::cam (MM84) cells were grown at 37°C to log phase, whereas topA20::Tn10 ΔtopB gyrB(Ts)/pSK760 (VU422), topA20::Tn10 ΔtopB gyrB(Ts)/pSK762c (VU425), topA20::Tn10 ΔtopB dnaT18::aph gyrB(Ts) (VU441), Δ(topA cysB) gyrB(Ts)/pEM001 (VU294), Δ(topA cysB) gyrB(Ts)/pEM003 (VU296), topA20::Tn10 gyrB(Ts) (RFM480), ΔtopB761::kan gyrB(Ts) (VU403), Δ(topA cysB) gyrB(Ts) ΔrecA306 srlR301::Tn10 (SB265) and Δ(topA cysB) ΔtopB::kan gyrB(Ts) ΔrecA306 srlR301::Tn10 (VU243) were grown at 30°C to log phase. Genomic DNA was extracted and qPCR was performed as described in Materials and Methods.

Fig 5. Dot blots with S9.6 antibodies to detect DNA:RNA hybrids in E. coli rnhA, topA and topA topB null mutants.

Wild-type (RFM443) and rnhA::cam (MM84) cells were grown at 37°C to log phase, whereas topA20::Tn10 ΔtopB gyrB(Ts)/pSK760 (VU422), topA20::Tn10 ΔtopB gyrB(Ts)/pSK762c (VU425) and topA20::Tn10 gyrB(Ts) (RFM480) cells were grown at 30°C to log phase. RFM480 downshift is topA20::Tn10 gyrB(Ts) cells grown at 37°C to log phase and transferred for 45 min. at 30°C. Genomic DNA was extracted and dot blotting was performed as described in Materials and Methods. RNase HI–and + mean that the genomic DNA samples were respectively not treated and treated with RNase HI as described in Materials and Methods.

Fig 6. qseC/lepA ratio in E. coli topA and topA topB null mutants.

In (a) wild-type (RFM443) cells were grown at 37°C to log phase, whereas Δ(topA cysB) gyrB(Ts) (RFM475), ΔtopB761::kan gyrB(Ts) (VU403), topA20::Tn10 gyrB(Ts) (RFM480), topA20::Tn10 ΔtopB gyrB(Ts) (VU421), topA20::Tn10 ΔtopB gyrB(Ts)/pSK760 (VU422), topA20::Tn10 ΔtopB gyrB(Ts)/pSK762c (VU425), Δ(topA cysB) ΔtopB::kan gyrB(Ts)/pSK760 (VU306), Δ(topA cysB) ΔtopB::kan gyrB(Ts)/pSK762c (VU333) topA20::Tn10 ΔtopB dnaT18::aph gyrB(Ts) (VU441) and Δ(topA cysB) ΔtopB::kan gyrB(Ts) ΔrecA306 srlR301::Tn10 (VU243) were grown at 30°C to log phase. Genomic DNA was extracted and qPCR was performed as described in Materials and Methods. In (b) ΔtopB::kan transductants of SS12 (Δ(topA cysB) gyrB(Ts)/pET11-parEC; strains JB37 and JB38)) and RFM475 (Δ(topA cysB) gyrB(Ts); strain JB40) were grown at 30°C to log phase and genomic DNA was extracted and qPCR was performed as described in Materials and Methods.