Distribution of DNA gyrase cleavage sites across the Streptococcus pneumoniae genome: relation to transcription and methylation at GATC sites.

Abstract

In bacteria, DNA gyrase plays a key role in cellular processes by affecting supercoiling. However, how gyrase localizes on the chromosome to affect supercoiling is unclear. Here, we interrogate the genome-wide distribution of gyrase in Streptococcus pneumoniae by mapping its cleavage sites in the presence of fluoroquinolones. Fluoroquinolones induce the formation of DNA-fluoroquinolone-topoisomerase complexes and generate detrimental double-stranded DNA breaks. Using a chromatin immunoprecipitation-sequencing-based method, we localize gyrase cleavage sites with single-nucleotide resolution. A total of 1517 sites were detected; the majority (92.7%) were located within protein-coding genes and were associated with high-level transcription. This trend was maintained at the level of transcriptional topological domains, where the most transcribed genes exhibited the highest frequency of cleavage sites, while the least transcribed genes exhibited the lowest frequency. We identified a four-base motif, GxxC, in 79% of the sites. The most frequent sequence was GATC (21.2% of all cleavage sites). GATC is the target of three pneumococcal restriction systems: DpnI, DpnII, and DpnIII. We demonstrated that methylation at GATC by the DpnII (GAmeTC) or DpnIII (GATCme) systems decreased gyrase cleavage and supercoiling activity. We hypothesized that strains with methylated GATC would be more resistant to subinhibitory concentrations of fluoroquinolones. We indeed demonstrated that gyrase mutants appeared more frequently in strains with GATC methylation (DpnII and DpnIII) than in strains without methylation (DpnI).This is significant for the evolution of fluoroquinolone resistance in S. pneumoniae, as the three Dpn phenotypes are present in the population of clinical isolates.