Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across theEscherichia coligenome

Abstract

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling

“…Based on the latter data, our modeling may thus be applicable to most moderately transcribed genes in the globally underwound bacterial genome, where the key regulatory step occurs at the initiation step and elongation does not generate comparably drastic mechanical constraints. This model might thus be inaccurate for very strong promoters like those of ribosomal RNAs (19), where the latter constraints may be handled by specific mechanisms disregarded here, such as a particular 3D organisation of the domain and high-affinity gyrase downstream binding sites (38).…”

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35–38 Amino Acids of GyrA

“…The importance of the arm sequence effect was unknown at the time of the early gyrase binding experiments, and BIMEs clearly have other functions, including the role of being loading sites on transcribed RNA for the transcription termination factor Rho [82]. Experiments using Next Generation Sequencing analysis of covalent GyrA-DNA complexes trapped by SDS lysis were recently reported for E. coli [83]. A similar study focused on Salmonella Typhimurium could answer many questions about species evolution and show precisely where gyrase binding is most critical for the class of highly transcribed operons.…”

Coherent Domains of Transcription Coordinate Gene Expression During Bacterial Growth and Adaptation

“…Activation of these strong transcriptons requires high negative superhelicity, supplied on the one hand by DNA gyrase, demonstrating a decreasing gradient of binding sites from OriC to Ter [19,21] and on the other hand, by increased production of negative supercoils in the wake of both the translocating replisomes and numerous RNAP molecules engaged in the transcription of stable RNA operons, all of which are oriented towards the Ter [56][57][58]. Notably, these powerful DNA translocases also increase the positive superhelicity ahead of them, which can be counterbalanced by increased gyrase activity observed downstream of the highly transcribed operons [59,60]. Although direct experimental evidence is still lacking, all these effects are thought to lead to higher negative superhelicity in the chromosomal OriC end compared to the Ter end [61], thus facilitating the utilization of thermodynamically stable (G/C-richer) DNA sequences for transcription.…”