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.

Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions.DOI: http://dx.doi.org/10.7554/eLife.25624.001

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Summary Nucleoid‐associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine‐ and arginine‐rich carboxy‐terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H‐NS. Lsr2 binds to and regulates expression of A/T‐rich portions of the otherwise G/C‐rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy‐terminal domain of HU and the acidic amino‐terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU‐Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.

The persistence of Mycobacterium tuberculosis ( Mtb ) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence . However, the mechanisms involved in NO sensing and reorganizing Mtb 's physiology are not fully understood. Since NO damages iron-sulfur (Fe–S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe–S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR ( Rv1460 ) senses NO via its 4Fe–4S cluster and promotes persistence of Mtb by mobilizing the Fe–S cluster biogenesis system; suf operon ( Rv1460-Rv1466 ). Analysis of anaerobically purified SufR by UV–visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe–4S cluster. Atmospheric O 2 and H 2 O 2 gradually degrade the 4Fe–4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe–4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe–S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR . Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe–S cluster metabolism and bioenergetics.