Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase. The generation of DNA topological constraint is intrinsic to transcription. Although in vitro studies indicated DNA gyrase and topoisomerase I action in the removal of excess supercoils, ahead and behind the transcribing RNA polymerase, in vivo recruitment and interaction of both topoisomerases with the genome has not been investigated. Using advanced sequencing, we have mapped the genome-wide footprints of topoisomerase I and DNA gyrase along with RNAP in deadly pathogen, Mycobacterium tuberculosis. We show that in vivo distribution of topoisomerases is guided by active transcription and both the enzymes co-occupy active transcription units. We establish their interaction with the regions of genome having propensity to accumulate negative and positive supercoiled domains, validating their role in managing the twin supercoiled domains.

Mycobacterium tuberculosis has evolved several mechanisms to counter host defense arsenals for its proliferation. Here we report that M. tuberculosis employs a multi-pronged approach to modify host epigenetic machinery for its survival. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating histone H3K79 in a non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing DOT1L-mediated nucleosomally added H3K79me3 mark on pro-inflammatory response genes. Consequent inhibition of caspase-8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3, and TMTC1, enabling the pathogen to overcome host inflammatory and oxidative responses. We provide the structural basis for differential methylation of H3K79 by Rv2067c and DOT1L. The structures of Rv2067c and DOT1L explain how their action on H3K79 is spatially and temporally separated, enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signaling.

DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.

Elizabethkingia anophelis has now emerged as an opportunistic human pathogen. However, its mechanisms of transmission remain unexplained. Comparative genomic (CG) analysis of E. anopheles endophthalmitis strain surprisingly found from an eye infection patient with twenty-five other E. anophelis genomes revealed its potential to participate in horizontal gene transfer. CG analysis revealed that the study isolate has an open pan genome and has undergone extensive gene rearrangements. We demonstrate that the strain is naturally competent, hitherto not reported in any members of Elizabethkingia. Presence of competence related genes, mobile genetic elements, Type IV, VI secretory systems and a unique virulence factor arylsulfatase suggests a different lineage of the strain. Deciphering the genome of E. anophelis having a reservoir of antibiotic resistance genes and virulence factors associated with diverse human infections may open up avenues to deal with the myriad of its human infections and devise strategies to combat the pathogen.