Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli.

The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.

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

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.

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

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.

Interplay between type 1A topoisomerases and gyrase in chromosome segregation in Escherichia coli.

Escherichia coli possesses two type 1A topoisomerases, Topo I (topA) and Topo III (topB). Topo I relaxes excess negative supercoiling, and topA mutants can grow only in the presence of compensatory mechanisms, such as gyrase mutations. topB mutants grow as well as wild-type cells. In vitro, Topo III, but not Topo I, can efficiently decatenate DNA during replication. However, in vivo, a chromosome segregation defect is seen only when both type 1A topoisomerases are absent. Here we present experimental evidence for an interplay between gyrase and type 1A topoisomerases in chromosome segregation. We found that both the growth defect and the Par(-) phenotypes of a gyrB(Ts) mutant at nonpermissive temperatures were significantly corrected by deleting topA, but only when topB was present. Overproducing Topo IV, the major cellular decatenase, could not substitute for topB. We also show that overproducing Topo III at a very high level could suppress the Par(-) phenotype. We previously found that the growth and chromosome segregation defects of a triple topA rnhA gyrB(Ts) mutant in which gyrase supercoiling activity was strongly inhibited could be corrected by overproducing Topo III (V. Usongo, F. Nolent, P. Sanscartier, C. Tanguay, S. Broccoli, I. Baaklini, K. Drlica, and M. Drolet, Mol. Microbiol. 69:968-981, 2008). We show here that this overproduction could be bypassed by substituting the gyrB(Ts) allele for a gyrB(+) one or by growing cells in a minimal medium, conditions that reduced both topA- and rnhA-dependent unregulated replication. Altogether, our data point to a role for Topo III in chromosome segregation when gyrase is inefficient and suggest that Topo I plays an indirect role via supercoiling regulation.

R-Loop Detection in Bacteria.

Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction. However, direct evidence for R-loop formation on chromosomal DNA from E. coli cells was only obtained recently by using the S9.6 antibody to detect RNA-DNA hybrids in dot-blot experiments. Here, we present a protocol for such experiments with a special emphasis on the procedure used for bacterial genomic DNA extraction and preparation including treatment with appropriate ribonucleases to eliminate RNA-RNA hybrids (that are also recognized by S9.6) as well as single-stranded RNA (ssRNA), in order to obtain a signal that is specific to stable RNA-DNA hybrids generated. Furthermore, we recommend that the results of such experiments be correlated with RNase HI-sensitive phenotypes.

Chiropractic management of patients post-disc arthroplasty: eight case reports.

BACKGROUND: When conservative therapies for low back pain (LBP) are not effective, elective surgery may be proposed to these patients. Over the last 20 years, a new technology, disc replacement, has become increasingly popular because it is believed to maintain or restore the integrity of spinal movement and minimize the side-effects compared to fusion. Although disc replacement may relieve a patient from pain and related disability, soreness and stiffness of the lumbopelvic region seem to be common aftermaths of the surgery. This prospective case series was undertaken to identify and describe potential adverse events of lumbar spinal manipulation, a common therapy for low back pain, in a group of patients with symptoms after disc prostheses. CASES PRESENTATION: Eight patients who underwent lumbar spine total disc replacement were referred by an orthopaedic surgeon for chiropractic treatments. These patients had 1 or 2 total lumbar disc replacements and were considered stable according to the surgical protocol but presented persistent, post-surgical, non-specific LBP or pelvic pain. They were treated with lumbar spine side posture manipulations only and received 8 to 10 chiropractic treatments based on the clinical evolution and the chiropractor's judgment. Outcome measures included benign, self-limiting, and serious adverse events after low back spinal manipulative therapy. The Oswestry Disability Index, a pain scale and the fear avoidance belief questionnaire were administered to respectively assess disability, pain and fear avoidance belief about work and physical activity. This prospective case series comprised 8 patients who all had at least 1 total disc replacement at the L4/L5 or L5/S1 level and described persistent post-surgical LBP interfering with their daily activities. Commonly-reported side-effects of a benign nature included increased pain and/or stiffness of short duration in nearly half of the chiropractic treatment period. No major or irreversible complication was noted. CONCLUSIONS: During the short treatment period, no major complication was encountered by the patients. Moreover, the benign side-effects reported after lumbar spine manipulation were similar in nature and duration to those frequently experienced by the general population.

Supercoiling, R-loops, Replication and the Functions of Bacterial Type 1A Topoisomerases.

Type 1A topoisomerases (topos) are the only topos that bind single-stranded DNA and the only ones found in all cells of the three domains of life. Two subfamilies, topo I and topo III, are present in bacteria. Topo I, found in all of them, relaxes negative supercoiling, while topo III acts as a decatenase in replication. However, recent results suggest that they can also act as back-up for each other. Because they are ubiquitous, type 1A enzymes are expected to be essential for cell viability. Single topA (topo I) and topB (topo III) null mutants of Escherichia coli are viable, but for topA only with compensatory mutations. Double topA topB null mutants were initially believed to be non-viable. However, in two independent studies, results of next generation sequencing (NGS) have recently shown that double topA topB null mutants of Bacillus subtilis and E. coli are viable when they carry parC parE gene amplifications. These genes encode the two subunits of topo IV, the main cellular decatenase. Here, we discuss the essential functions of bacterial type 1A topos in the context of this observation and new results showing their involvement in preventing unregulated replication from R-loops.

Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation.

Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought.

R-loop-dependent replication and genomic instability in bacteria.

DNA replication, the faithful copying of genetic material, must be tightly regulated to produce daughter cells with intact copies of the chromosome(s). This regulated replication is initiated by binding of specific proteins at replication origins, such as DnaA to oriC in bacteria. However, unregulated replication can sometimes be initiated at other sites, which can threaten genomic stability. One of the first systems of unregulated replication to be described is the one activated in Escherichia coli mutants lacking RNase HI (rnhA). In fact, rnhA mutants can replicate their chromosomes in a DnaA- and oriC-independent process. Because this replication occurs in cells lacking RNase HI, it is proposed that RNA from R-loops is used as a DNA polymerase primer. Replication from R-loops has recently attracted increased attention due to the advent of DNA:RNA hybrid immunoprecipitation coupled with high-throughput DNA sequencing that revealed the high prevalence of R-loop formation in many organisms, and the demonstration that R-loops can severely threaten genomic stability. Although R-loops have been linked to genomic instability mostly via replication stress, evidence of their toxic effects via unregulated replication has also been presented. Replication from R-loops may also beneficially trigger stress-induced mutagenesis (SIM) that assists bacterial adaptation to stress. Here, we describe the cis- and trans-acting elements involved in R-loop-dependent replication in bacteria, with an emphasis on new data obtained with type 1A topoisomerase mutants and new available technologies. Furthermore, we discuss about the mechanism(s) by which R-loops can reshape the genome with both negative and positive outcomes.

Hypernegative supercoiling inhibits growth by causing RNA degradation.

Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.

Detection of oriC-Independent Replication in Escherichia coli Cells.

In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2'-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor® 488 dye is conjugated to EdU by the click-iT® reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy.

Effects of DNA supercoiling and topoisomerases on the expression of genes coding for F165(1), a P-like fimbriae.

Pathogenic Escherichia coli 4787 (O115:KV165) causes septicemia in pigs and expresses the fimbriae F165(1) encoded by the foo operon that belongs to the P fimbrial family. fooI and fooB, encoding specific foo regulators, are divergently transcribed; their intergenic region is responsible for the regulation of foo expression. The role of global and local supercoiling (transcription-induced supercoiling within the intergenic region) on the regulation of foo expression was investigated. Expression of fooB was significantly altered when global negative supercoiling was reduced by a mutation that decreases DNA gyrase activity. Deletion of the topA gene, encoding for topoisomerase I that relaxes local negative supercoiling, further reduced fooB expression. This suggests that both global and local supercoiling can significantly affect fooB expression. Moreover, FooI, a positive regulator of fooB expression, has no effect on fooB expression in the topA null mutant. This study showed that divergent transcription from a strong promoter can significantly enhance fooB expression and compensate for the absence of FooI in a wild-type strain.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA.

Constitutive stable DNA replication in Escherichia coli cells lacking type 1A topoisomerase activity.

Type 1A topoisomerases (topos) are ubiquitous enzymes involved in supercoiling regulation and in the maintenance of genome stability. Escherichia coli possesses two type 1A enzymes, topo I (topA) and topo III (topB). Cells lacking both enzymes form very long filaments and have severe chromosome segregation and growth defects. We previously found that RNase HI overproduction or a dnaT::aph mutation could significantly correct these phenotypes. This leads us to hypothesize that they were related to unregulated replication originating from R-loops, i.e. constitutive stable DNA replication (cSDR). cSDR, first observed in rnhA (RNase HI) mutants, is characterized by its persistence for several hours following protein synthesis inhibition and by its requirement for primosome components, including DnaT. Here, to visualize and measure cSDR, the incorporation of the nucleotide analog ethynyl deoxyuridine (EdU) during replication in E. coli cells pre-treated with protein synthesis inhibitors, was revealed by "click" labeling with Alexa Fluor(®) 488 in fixed cells, and flow cytometry analysis. cSDR was detected in rnhA mutants, but not in wild-type strains, and the number of cells undergoing cSDR was significantly reduced by the introduction of the dnaT::aph mutation. cSDR was also found in topA, double topA topB but not in topB null cells. This result is consistent with the established function of topo I in the inhibition of R-loop formation. Moreover, our finding that topB rnhA mutants are perfectly viable demonstrates that topo III is not uniquely required during cSDR. Thus, either topo I or III can provide the type 1A topo activity that is specifically required during cSDR to allow chromosome segregation.

The problem of hypernegative supercoiling and R-loop formation in transcription.

DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I.

RNase H overproduction allows the expression of stress-induced genes in the absence of topoisomerase I.

Induction of stress proteins in response to heat shock was found to be reduced significantly in Escherichia coli with DeltatopA mutation. RNase H overexpression in the DeltatopA mutant partially restored the sigma(32)-dependent induction of stress genes in response to high temperature and ethanol. The presence of overexpressed RNase H also improved the survival rate of the DeltatopA mutant after high temperature and oxidative challenges. Topoisomerase I is likely required during stress response for preventing accumulation of transcription-driven hypernegative supercoiling and R-loop formation at induced stress genes loci.

Efficacy of preventive spinal manipulation for chronic low-back pain and related disabilities: a preliminary study.

OBJECTIVE: To document the potential role of maintenance chiropractic spinal manipulation to reduce overall pain and disability levels associated with chronic low-back conditions after an initial phase of intensive chiropractic treatments. METHODS: Thirty patients with chronic nonspecific low-back pain were separated into 2 groups. The first group received 12 treatments in an intensive 1-month period but received no treatment in a subsequent 9-month period. For this group, a 4-week period preceding the initial phase of treatment was used as a control period to examine the sole effect of time on pain and disability levels. The second group received 12 treatments in an intensive 1-month period and also received maintenance spinal manipulation every 3 weeks for a 9-month follow-up period. Pain and disability levels were evaluated with a visual analog scale and a modified Oswestry questionnaire, respectively. RESULTS: The 1-month control period did not modify the pain and disability levels. For both groups, the pain and disability levels decreased after the intensive phase of treatments. Both groups maintained their pain scores at levels similar to the postintensive treatments throughout the follow-up period. For the disability scores, however, only the group that was given spinal manipulations during the follow-up period maintained their postintensive treatment scores. The disability scores of the other group went back to their pretreatment levels. CONCLUSIONS: Intensive spinal manipulation is effective for the treatment of chronic low back pain. This experiment suggests that maintenance spinal manipulations after intensive manipulative care may be beneficial to patients to maintain subjective postintensive treatment disability levels. Future studies, however, are needed to confirm the finding in a larger group of patients with chronic low-back pain.

Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology.

It has been known for a long time that supercoiling can affect gene expression at the level of promoter activity. Moreover, the results of a genome-wide analysis have recently led to the proposal that supercoiling could play a role in the regulation of gene expression at this level by acting as a second messenger, relaying environmental signals to regulatory networks. Although evidence is lacking for a regulatory role of supercoiling following transcription initiation, recent results from both yeast and bacteria suggest that the effect of supercoiling on gene expression can be considerably more dramatic after this initiation step. Transcription-induced supercoiling and its associated R-loops seem to be involved in this effect. In this context, one major function of topoisomerases would be to prevent the generation of excess negative supercoiling by transcription elongation, to inhibit R-loop formation and allow gene expression. This function would be especially evident when substantial and rapid gene expression is required for stress resistance, and it may explain, at least in part, why topoisomerase I synthesis is directed from stress-induced promoters in Escherichia coli. Growth inhibition mediated by excess negative supercoiling might be related to this interplay between transcription elongation and supercoiling.

Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli.

Acid resistance (AR) in Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (the GAD system) requires the glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (the ARG system) requires the arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2.5 treatment by 10-fold to >100-fold. The RpoS and GAD systems were both affected by the topA mutation, but the ARG system of AR was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5.5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I might have a direct influence on the transcription of AR genes. This influence does not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of AR caused by the topA mutation. The effect of the topA mutation could be suppressed by an hns mutation, so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression, in addition to its influence on RpoS-dependent transcription.

Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation.

Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R-loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription-induced supercoiling. Effects on R-loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R-loop-dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R-loop-dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R-loop-dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R-loop-dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R-loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady-state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription-translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.