Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces ofgenetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression ofRNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.DNA topoisomerase (TOPO) I, originally known as o, is the major DNA relaxing activity in Escherichia coli (1). The gene encoding E. coli DNA TOPO I topA was localized to the cys-trp region of the chromosome (2, 3). Deletions that remove both cysB and topA were identified in E. coli strains and it was first assumed that topA was not an essential gene (3). A more careful analysis showed that a deletion of the topA gene could only be inherited in strains that contained a second mutation that would compensate for the loss of topA (4, 5). These studies identified mutations in both gyrA and gyrB that could compensate for the loss of topA and demonstrated that these mutations result in reduced global DNA supercoiling. This lead to a picture in which a "global balance" of DNA supercoiling is essential and controlled by the competing activities of DNA gyrase and TOPO I.In vitro DNA TOPO I does not relax negatively supercoiled DNA to completion (1). Other in vitro studies show that the specificity for negatively supercoiled DNA derives from a requirement of a short single-stranded DNA region as part of the enzyme-DNA complex (6). Since negative DNA supercoiling favors the unpairing of DNA strands, DNA molecules with high negative superhelical density will be a better substrate for DNA TOPO I. Some in vivo studies also suggest that DNA TOPO I does not relax DNA efficiently: in one study a topA mutation had little effect on the rate of in vivo DNA relaxation after the inhibition of DNA gyrase by coumermycin (7); and in a second in vivo study, DNA TOPO I produced only slow and partial DNA relaxation (8). However, a defect in DNA TOPO I can cause an in vivo increase in the level of negative supercoiling (5). Thus, these results suggest that DNA TOPO I functions to prevent DNA supercoiling from reaching an unacceptably high level.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3526Hypernegatively supercoiled DNA can, under certain circumstances, be produced during transcription (9, 10). In...

SummaryIt 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.

The nascent RNA is normally displaced during the process of transcription to be translated or to participate in the process of translation. Shortly after the discovery that cellular DNA can be negatively supercoiled, it was suggested that the favorable free energy of such supercoiling should maintain the base pairing between the nascent RNA and the template DNA strand, and consequently, should interfere with the process of RNA displacement. Indeed, a direct correlation between the level of negative supercoiling and the length of the RNA-DNA hybrid (or R-loop, the template strand is paired with RNA, leaving the nontemplate strand unpaired) after transcription with Escherichia coli RNA polymerase was found (1). The formation of such hybrids was later shown to be due to the denaturing of transcribing RNA polymerases, and hence to the use of protein denaturing agents to stop the transcription reactions (2). These experiments have also demonstrated that the RNA polymerase possesses a putative "separator" function allowing the nascent RNA to be displaced as transcription proceeds, and therefore a function that counteracts the favorable free energy of negative supercoiling for RNA-DNA hybrid formation. In agreement with this notion are the results from several experiments revealing that the 9 -12-base pair RNA-DNA hybrid within the RNA polymerase is positioned very close to the downstream edge of the 18-base pair open transcription bubble (3). Thus, extensive R-loop formation originating from the short hybrid within the transcription bubble does not normally occur during transcription.However, the results of several in vitro and in vivo experiments have clearly shown that extensive R-loops can form during transcription on negatively supercoiled templates, and that both the formation and the length of such structures is modulated by DNA topoisomerases (4 -7). These topoisomerases are, DNA gyrase, responsible for the introduction of negative supercoiling, and DNA topoisomerase I, responsible for the relaxation of negative supercoiling (reviewed in Ref. 8). Indeed, in one study (5), overproduction of RNase H, an enzyme degrading the RNA moiety of an R-loop, was shown to partially correct the growth defect of topA null mutants. In another series of experiments, R-loop formation during transcription of a portion of the rrnB operon, encoding for rRNAs, was shown to occur both in vitro and in vivo in the absence of DNA topoisomerase I (6, 7). How these results can be pieced together with the early observations described above is still unknown. One way to reconcile all these observations is to consider that the R-loop does not originate from the transcription bubble but is initiated by the reannealing of a portion of the nascent RNA with a complementary DNA template region behind the moving RNA polymerase. This, of course, requires that the nascent RNA be free and therefore not bound by ribosomes and that the corresponding DNA region behind the moving RNA polymerase be opened. DNA opening behind the moving RNA polymerase can b...

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...

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.