Rewired Downregulation of DNA Gyrase Impacts Cell Division, Expression of Topology Modulators, and Transcription in Mycobacterium smegmatis

Accepted Manuscript Rewired Downregulation of DNA Gyrase Impacts Cell Division, Expression of Topology Modulators, and Transcription in Mycobacterium smegmatis

Sarmistha Guha, Shubha Udupa, Wareed Ahmed, Valakunja Nagaraja PII: DOI: Reference:

S0022-2836(18)30258-4 doi:10.1016/j.jmb.2018.10.001 YJMBI 65886

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

15 April 2018 22 September 2018 2 October 2018

Please cite this article as: Sarmistha Guha, Shubha Udupa, Wareed Ahmed, Valakunja Nagaraja , Rewired Downregulation of DNA Gyrase Impacts Cell Division, Expression of Topology Modulators, and Transcription in Mycobacterium smegmatis. Yjmbi (2018), doi:10.1016/j.jmb.2018.10.001

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ACCEPTED MANUSCRIPT Rewired downregulation of DNA gyrase impacts cell division, expression of topology modulators, and transcription in Mycobacterium smegmatis Sarmistha Guhaa, Shubha Udupaa, Wareed Ahmeda and Valakunja Nagarajaa,b,c

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a) Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore

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560012, India

b) Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560012, India

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c) To whom correspondence should be addressed: V. Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-

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560012, India. Tel: +91-80-23600668, Fax: +91-80-23602697

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E-mail: [email protected]

DNA

supercoiling,

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Keywords: Conditional

knockdown,

Relaxation-stimulated

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supercoiling-sensitive promoters, RNA polymerase. Abbreviations

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ATc - Anhydrotetracycline cKD - Conditional knockdown Gyr - Gyrase Mtb - Mycobacterium tuberculosis NAPs - Nucleoid-Associated Proteins RNAP - RNA Polymerase RST - Relaxation-Stimulated Transcription ORF - Open Reading Frame SST - Supercoiling Sensitive Transcription topo I - Topoisomerase I topo IV - Topoisomerase IV TUs - Transcription units

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transcription,

ACCEPTED MANUSCRIPT Abstract: DNA gyrase, essential for DNA replication and transcription, has traditionally been studied in vivo by treatments that inhibit the enzyme activity. Due to its indispensable function, gyrA and gyrB deletions cannot be generated. The coumarin

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inhibitors of gyrase induce the supercoiling-sensitive gyrase promoter by a mechanism

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termed relaxation-stimulated transcription (RST). Hence, to study the effect of sustained

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reduction in gyrase levels, a conditional-knockdown strain was generated in Mycobacterium smegmatis such that gyrase expression was controlled by a

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supercoiling non-responsive regulatory circuit. Decreasing intracellular gyrase protein

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levels beyond 50% affected cell growth. Reduced gyrase levels in the reprogrammed gyr operon caused chromosome relaxation, diffuse nucleoid structure, cell elongation,

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and altered gene expression. The key cell division protein, ftsZ, was severely reduced

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in the elongated cells, indicating a link between gyrase and cell division. Low levels of gyrase resulted in low compensatory expression of topoisomerase I and elevated

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expression of topology modulators hupB and lsr2. Altered supercoiling due to gyrase

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depletion caused corresponding changes in the RNA polymerase (RNAP) density on transcription units leading to their altered transcription. The enhanced susceptibility of

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the knockdown strain to anti-tubercular drugs suggests its utility for screening new molecules that may act synergistically with gyrase inhibitors.

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ACCEPTED MANUSCRIPT INTRODUCTION In eubacteria, the concerted action of topoisomerases maintains a set level of DNA supercoiling through the action of gyrase and the supercoil relaxing activities of topoisomerase I (topo I) and topoisomerase IV (topo IV) [1], [ 2], [ 3], [ 4]. Topo I relaxes

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excess negative supercoils generated by the replication and transcription machinery,

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while DNA gyrase introduces negative supercoiling in an ATP-dependent manner to

controlling the transcription

of

topoisomerase

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maintain optimal negative supercoiling [5]. Supercoil maintenance is regulated by genes via

relaxation-stimulated

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transcription (RST) [6], [ 7] and supercoiling-sensitive transcription (SST) [6], [ 8], [ 9], [

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10]. DNA supercoiling is also controlled by cellular energetics. The metabolic state can alter the [ATP]/[ADP] ratio in the cell [11], [ 12], which in turn influences DNA gyrase

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activity resulting in a change in the superhelical density of the chromosome [12]. Other

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contributors to the maintenance of DNA supercoiling are the nucleoid-associated proteins (NAPs). NAPs are abundant, small basic proteins that bind, bend, and bridge

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DNA, and modulate transcription by constraining supercoils [13]. Previous studies

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demonstrated that histone-like protein (HU), a type of NAP, influences supercoiling by its ability to wrap DNA, and a 12% reduction in supercoiling was observed upon the

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removal of HU [12].

While a majority of bacterial species studied so far encode both DNA gyrase and topo IV, M. smegmatis lacks topo IV [14], [ 15]. Thus, DNA gyrase exhibits a dual enzymatic role carrying out activities of both DNA supercoiling and decatenation [16], [ 17]. The decatenation activity of mycobacterial DNA gyrase may have a role in DNA segregation [18]. However, the enzyme level required to regulate cellular activities in

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ACCEPTED MANUSCRIPT vivo is not known. Further, consequences of lowering the abundance of this essential enzyme on bacterial growth and physiology are not understood. Previous studies to understand the role of DNA gyrase in Escherichia coli relied on using inhibitors of DNA gyrase [6], [ 19], [ 20], [ 21], steady-state mutants [2], [ 22]

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and temperature-sensitive (ts) mutants [2], [ 23], [ 24]. DNA gyrase inhibitors result in

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chromosome relaxation, which, in turn also enhances transcription at gyrB and gyrA

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promoters in E. coli [6], [ 21], promoter of the gyrase operon in M. smegmatis, M. tuberculosis [7], [ 25], Streptomyces coelicolor [26] and Staphylococcus aureus [27].

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However, long-term treatment with the toxic inhibitors would compromise the growth of

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bacteria as well as create unexplored off-target effects that may complicate the inference. Moreover, intracellular functional gyrase molecules would be reduced for a

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limited duration only, as the consequent loss of supercoiling would increase gyrase

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expression by RST [6], [21]. Steady-state mutants exhibit either excessive or low levels of supercoiling and have growth rates similar to that of WT. Moreover, steady-state

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mutants are subjected to additional unknown mutations [1], [ 2], [ 22]. When gyrase ts

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mutants are used, non-permissive temperatures lower the activity of gyrase while the shift in temperature towards the permissive range restores the gyrase activity and

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supercoiling similar to that of the WT [23]. Thus, understanding of the contribution of gyrase in gene regulation has not been fully appreciated. One way to address the issues mentioned above is to construct a strain with minimal gyrase constitution wherein the gyrase regulation is rewired to be unresponsive to RST. The present study investigates how conditional knockdown of DNA gyrase influences the growth and physiology of mycobacteria. A TetR/Pip OFF repressible

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ACCEPTED MANUSCRIPT promoter system [28] was used wherein the gyrase operon is placed under the control of a supercoil-insensitive promoter and expressed in M. smegmatis. The effect of perturbation of supercoiling by reducing gyrase level on cell growth, chromosome architecture and transcription in M. smegmatis was explored. The strain exhibited

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growth defects, diffuse nucleoid, reduced supercoiling, increased drug susceptibility and

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Bacterial strains, growth media and plasmids

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MATERIALS AND METHODS

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altered expression of DNA topology modulators.

The strains and plasmids used in the study are listed in Table 1. Transformations

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were carried out with competent E. coli (DH5 alpha) or electrocompetent M. smegmatis.

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Wild-type [29] and gyrase-depleted strains were grown at 30°C in Middlebrook 7H9 broth (Difco) or on 7H10 agar plates (Difco), supplemented with 0.2% glycerol and

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0.05% Tween-80. The cells were plated on Middlebrook 7H10 agar plates. Antibiotics

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were added to the media at the following concentrations: streptomycin, 20 µg/ml; kanamycin, 50 µg/ml; hygromycin, 150 µg/ml (E. coli) or 50 µg/ml (M. smegmatis).

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Anhydrotetracycline (ATc) (Sigma-Aldrich) was added at final concentrations ranging

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from 100-400 ng/ml. The pJAM-Rv3852 plasmid [30] having full-length Rv3852 cloned into pJAM2 vector system [31] was introduced into the WT, and MsGyrCk strains by electroporation

and

kanamycin-resistant

colonies

were

selected

to

obtain

mc2155::pJAM-Rv3852 and MsGyrCk::pJAM-Rv3852. The E. coli lacZ PCR amplicon (3069 bp) was cloned into the pMIND vector [32] using PacI and SpeI restriction sites to obtain lacZ-pMIND. The recombinant plasmid is then electroporated into WT and M. smegmatis promoter replaced strains (MsPRS) to obtain mc2155::pMIND-lacZ and 5

ACCEPTED MANUSCRIPT MsGyrCk::pMIND-lacZ strains respectively. Strains harboring pMIND vector only served as control. Table 1: List of plasmids and strains used in the study Name

Type

Description

Reference

plasmid

Suicide vector containing Pptr promoter

[28]

pFRA42B

plasmid

Integrative plasmid

[28]

pFRA50-MsGyrB

plasmid

pJAM-Rv3852

plasmid

pMIND-lacZ

plasmid

pMV261

plasmid

966bp of GyrB NTD cloned downstream to Pptr in the pFRA50 suicide vector Rv3852 expressed under acetamide inducible promoter Full-length lacZ expressed under tetracycline-inducible promoter Mycobacterial replicating vector

mc2155 [29]

strain

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MsGyrCk

strain

mc2155::pJAM-Rv3852

strain

MsGyrCk::pJAM-Rv3852

strain

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ED strain

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MsPRS

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pFRA50

Wild-typeM. smegmatis strain M. smegmatis having gyrase under the Pptr promoter. WT cells were electroporated with the pFRA50-MsGyrB plasmid. Gyrase conditional knockdown in M. smegmatis. WT cells harboring pFRA50-MsGyrB and pFRA42B plasmids. mc2155 ectopically expressing Rv3852 under acetamide inducible promoter of pJAM2. MsGyrCk ectopically expressing Rv3852 under acetamide inducible promoter of pJAM2.

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This study

[30]

This study

[33] Laboratory stock This study

This study

This study

This study

ACCEPTED MANUSCRIPT strain

MsGyrCk::pMIND-lacZ

strain

mc2155 ectopically expressing lacZ under tetracycline-inducible promoter. MsGyrCk ectopically expressing lacZ under tetracycline-inducible promoter.

This study

This study

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mc2155::pMIND-lacZ

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Construction of gyrase conditional knockdown in M. smegmatis

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966 bp of the gyrB amino-terminal domain (NTD) was amplified from genomic

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DNA of M. smegmatis. The amplified fragments were digested with NsiI and NheI, cloned downstream of the Pptr promoter in the suicide plasmid pFRA50 linearized with

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the same enzymes [28] to obtain recombinant plasmid pFRA50-MsGyrB. M. smegmatis cells were electroporated with pFRA50-MsGyrB to replace the native gyrase promoter

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with the Pip-controlled promoter (Pptr). Recombinant colonies were selected on 7H10

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agar plates containing hygromycin (50 mg/ml). Integration of the plasmid via insertional duplication was confirmed by PCR (Figure S1). The integrative plasmid pFRA42B [28]

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was introduced in the recombinant strain (MsPRS) by bacterial transformation to obtain

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a gyrase conditional knockdown (MsGyrCk). Growth analysis

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Colonies of MsPRS and MsGyrCk were grown on Middlebrook 7H10 agar supplemented with 0.5% glycerol and incubated at 30°C. Exponential cultures of the gyrase conditional strain were grown at 30°C in Middlebrook 7H9 media supplemented with 0.5% glycerol and 0.05% Tween-80. MsGyrCk and WT cultures were treated with 100–600 ng anhydrotetracycline (ATc) per ml for the indicated periods of time. For measuring the growth of WT, MsPRS and the MsGyrCk, exponential-phase cultures

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ACCEPTED MANUSCRIPT (O.D.595 of 0.6) were taken and diluted further in 7H9 media. Growth profiles were obtained using the BioScreen growth curve analyzer and plots were generated using GraphPad Prism (version 5.0). EtBr uptake assay

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Cells were grown to mid-log phase (O.D.595 of 0.4) were treated with EtBr (0.5 µg

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ml−1) for 10 min. The EtBr-loaded cells were centrifuged at 4000 rpm and resuspended

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in EtBr-free phosphate buffered saline (PBS) containing 0.4 % glucose. After adjusting the O.D.595 to 0.4, aliquots of 200 µl were transferred to a 96-well plate, and

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fluorescence was measured in a fluorimeter (Tecan infinite pro200) using the 290 nm

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and 580 nm as the excitation and detection wavelengths, respectively. ATP detection assay

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The luminescence-based Bactiter-Glo System (Promega), which uses ATP

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indicator to measure viable cells, was used. Assay results were measured using the Tecan infinite pro200 to detect the ATP concentrations.

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Drug sensitivity assay

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WT and MsGyrCk cells were grown to an O.D.595 of 0.6 and then diluted to a final O.D.595 of 0.05 with the fresh 7H9 medium. The culture was then aliquoted into a 100-

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well growth plate. Broth dilution method was used to determine minimal inhibitory concentration (MIC) of the drugs tested. Serial dilutions of the drugs were added to the culture, and untreated culture was taken as a control. The growth was monitored at 595 nm with continuous shaking at 30°C in the Bioscreen growth analyzer.

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ACCEPTED MANUSCRIPT Biofilm, pellicle and motility assays Biofilm formation was assessed as described previously [30], [ 34]. Briefly, 100 ml of 7H9 medium supplemented with 0.5 % Casamino acids (casein hydrolysate) was used per well in a 96-well PVC microtiter dish. After inoculation, microtiter dishes were

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incubated for 48 and 96 hr for the WT and knockdown strain, respectively (to an O.D.595

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of 0.6). Pellicle formation was monitored by growing standing cultures of mycobacteria

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in Middlebrook 7H9 medium without Tween-80 at 30C. Motility assays were carried out

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as described previously [35]. Briefly, cells were cultured in 7H9 medium to midexponential phase (O.D.595 of 0.6) before spotting 2 ml aliquots onto motility medium

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consisting of 7H9 supplemented with 0.5% casamino acids and 0.2% glycerol, solidified with low-melting-point agarose (0.4%, w/v). The inoculated plates were incubated for

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24–96 hr at 30C.

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Plasmid isolation and chloroquine gel electrophoresis

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Plasmid pMV261 (4.4 kb) was used as a reporter to monitor the relative difference in the levels of DNA supercoiling between WT and gyrase-conditional strains.

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pMV261 was isolated using an alkaline lysis method [36]. In brief, pMV261 was

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electroporated in the WT, promoter-replaced and conditional knockdown (cKD) strains. The transformants were selected on 7H10 agar containing kanamycin. Cultures (O.D.595 of 0.6) harboring pMV261 were treated with 25 mg/ml lysozyme for 12 hr followed by lysis. The isolated plasmids were resolved on 1.2% agarose gel in Tris Borate EDTA (TBE) buffer in the presence of 1.5 µg/ml, 2.5 µg/ml and 10 µg/ml chloroquine at 1 V/cm [37]. The gels were washed with water and stained with ethidium bromide for 30 min. The topoisomers of the plasmid from each strain were visualized using a digital imaging

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ACCEPTED MANUSCRIPT system (Bio-Rad). The relative amounts of supercoiling in plasmid DNA isolated from each strain were assessed to indicate the difference in DNA supercoiling [2]. Immunoblot analysis Cells were harvested and lysed by sonication in buffer (20 mM Tris pH 8.0, 1 mM

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EDTA, 10% glycerol, 5 mM β‐mercaptoethanol, 1 mM phenyl methyl sulfonyl fluoride, 1

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mM benzamidine HCl and 1% Triton X‐100). Total protein was estimated by Bradford

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method [38]. Lysates were separated on 8% SDS-polyacrylamide gels (SDS-PAGE),

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transferred onto PVDF membranes and blocked with 2 % (w/v) Bovine Serum Albumin (BSA) in PBS containing 0.2% Tween prior to incubation with indicated primary

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antibodies. Membranes were then washed in PBST (0.2% Tween-20) three times and incubated with horseradish peroxidase-conjugated goat polyclonal anti-mouse IgG or

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goat polyclonal anti-rabbit IgG secondary antibodies (GE Amersham). Proteins were

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then visualized using a chemiluminescent substrate (Millipore).

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Determination of amounts of gyrase

To determine the gyrase protein concentration, immunoblot analysis was

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performed with anti-GyrA antibody to obtain standard curves using different concentrations of purified GyrA. The range where linearity exists between the protein

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concentration and the intensity of immunostaining was determined. Using appropriate volumes of lysates, the measurements of three independently prepared lysates was done and interpolated using linear regression. Using the total number of micrograms of protein per ml, the number of molecules of gyrase per microgram of protein can be calculated [39]. For determining colony forming units (CFUs), aliquots of log phase cultures (OD600nm = 0.5) of WT and MsGyrCk were serially diluted in 7H9 broth (10-fold

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ACCEPTED MANUSCRIPT dilutions). 100 µl of appropriate dilutions were then spread on 7H10 agar, and the plates were incubated at 30°C for 72 hr before CFU were counted. To minimize variability in CFU/ml, a conversion from microgram of protein per ml was used. By correlating the total protein concentration of gyrase in a cell with CFU/ml, the number of gyrase

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molecules per cell was determined.

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Microscopy

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Cells were plated on poly-L-lysine-coated coverslips in 24-well plates. Bacteria were allowed to settle for 30 min before gently decanting and adding 1 ml solution

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containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate

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buffer (pH 7.4) with 0.2 M sucrose. Samples were treated with 2% OsO4 in 0.1 M sodium cacodylate buffer for 2 hr at room temperature. After a series of sequential

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ethanol dehydrations (30, 50, 70, 95 and 100%), samples were dried under vacuum,

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gold sputter coated and imaged with a Quanta-200 scanning electron microscope. For fluorescence microscopy, cells were fixed by using 2% toluene and Triton X-

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100 (1%) at 4C overnight. Before staining of the DNA, the cells were washed,

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suspended in PBS (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and treated with lysozyme (2 mg/ml). The cells were stained with DAPI (0.5 mg/ml) for 15 min, and the

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samples were examined using a confocal microscope (Zeiss LSM 880) equipped with a 100X objective.

RNA isolation and qRT-PCR Cells were grown to O.D595 = 0.6, 200 ng/ml ATc was added to one part, and the other served as untreated control. After 6 hr of ATc treatment, RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer’s protocol, digested with

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ACCEPTED MANUSCRIPT DNase I (Fermentas) to remove traces of chromosomal DNA and tested for DNA contaminations using PCR with oligonucleotides complementary to M. smegmatis gyrA. The number of cDNA copies obtained after reverse transcription of 10 ng of total RNA extracted from cells was calculated for each of the genes that were used in this study.

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Two aliquots of total RNA from independent preparations were subjected to reverse

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transcription with antisense primers specific for each of the genes tested. RNA samples

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not treated with reverse transcriptase were used to measure chromosomal DNA

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contamination. The number of chromosome equivalents per l was calculated by taking the length of the M. smegmatis chromosome as 6.9 Mb. Samples containing a known

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amount of target copies were used to generate a standard curve. The amount of target DNA in a sample is interpolated from a standard curve run in parallel with genomic

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DNA. For all the genes tested, the samples subjected to reverse transcription contained

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a higher amount of template molecules than untreated samples. For studies using WT

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cells treated with novobiocin, qRT-PCR was carried out using the Ct method [40]. Chromatin Immunoprecipitation (ChIP- RT PCR)

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ChIP with M. smegmatis cultures grown to mid-log phase was carried out as

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described previously [41]. Briefly, formaldehyde cross-linked cells were sonicated to shear the DNA to an average size of 150-500 bp using Diagenode bioruptor. 1 ml of each sample was incubated with anti-RpoC and anti-GyrA antibodies, and an aliquot of the samples without antibody processed similarly served as negative control (Mock-IP). Protein-DNA complexes were immunoprecipitated, and DNA was purified. ChIP DNA was subjected to qRT-PCR analysis to determine the enrichment of RNAP on target DNA in the IP sample over the mock-IP (without antibody).

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ACCEPTED MANUSCRIPT RESULTS: Construction and characterization of a gyrase conditional knockdown In M. smegmatis, the gyrB and gyrA genes are organized as an operon [7]. Pgyr, the sole promoter of the operon, located upstream of gyrB is subjected to RST upon

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novobiocin treatment [7]. To circumvent this autoregulatory supercoiling-sensitive

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circuit, downregulation of the gyr operon was achieved by placing the operon under the

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negative transcriptional control of the pip-dependent promoter, Pptr (Figure 1 A) [28]. Successful replacement of the native promoter by single-site integration was confirmed

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by detection of a 966bp PCR-amplified product by agarose gel electrophoresis

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(Supplementary Figure S1). The resulting strain (MsPRS) was transformed into the integrative pFRA42B plasmid [28] to obtain strain MsGyrCk. Gyrase levels in the

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promoter-replaced strain (MsPRS) were found to be ~40% lower when compared to WT

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(Figure 1B), indicating that the canonical gyrase promoter is stronger than Pptr. However, the addition of ATc resulted in the induction of pristinamycin repressor, and

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to WT (Figure 1B).

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repression of Pptr caused a further reduction in gyrase level beyond 50% as compared

Given the crucial role of DNA gyrase in bacteria, its downregulation could impact

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the growth of M. smegmatis. Notably, the cultures of MsGyrCK exhibited temperature sensitivity (failure to grow at 37°C). On lowering the temperature to 30°C, the strain exhibited growth with a prolonged lag phase (about 16-18 hr; Figure 2A). The reduction in growth was found to be dose-dependent, i.e., increasing ATc concentration (100 to 600 ng/ml) led to a prolonged growth arrest (Figure 2B, Supplementary Figure S2). With the periodic addition of ATc, the cells not only exhibited a prolonged lag phase but

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ACCEPTED MANUSCRIPT grew slower than WT due to the continuous reduction in gyrase levels (Figure 2C). When the growth defect was further analyzed on solid agar, MsGyrCk required 8 days to form colonies compared to 4 days for WT. Moreover, MsGyrCk was unable to grow in higher cell dilutions compared to the WT (Figure 2D, Supplementary Figure S2).

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These results confirm the essentiality of DNA gyrase in the growth of M. smegmatis.

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Next, to determine the minimum gyrase level required for cell growth, the number

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of gyrase molecules was estimated in the MsGyrCk using semi-quantitative western blotting and colony forming unit (CFU) analysis under conditions where the addition of

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ATc repressed gyrase levels until viable colonies were obtained on agar plates. The

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addition of ATc to a dose of 500 ng/ml ATc led to a reduction of gyrase level by ~3.5fold (Figure 2E) and yielded minimum viable colonies (Supplementary Figure S3).

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With a further increased dose of ATc (600 ng/ml), no colonies appeared, indicating that

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gyrase expression reduced to a level where cells had insufficient gyrase molecules for cell growth. The average number of gyrase molecules during exponential phase (O.D. =

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0.5) of WT cells was around 1250 ± 100 (CFU/ml = 88X107), whereas the number was

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reduced to around 700 ± 100 molecules (CFU/ml = 26X107) in the gyrase conditional knockdown strain upon treatment with 500 ng of ATc. These results suggest that a

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minimal gyrase level is necessary for cell sustenance, and a reduction in the number of molecules beyond the threshold level can be detrimental for bacterial growth. Gyrase knockdown leads to altered cell morphology and nucleoid architecture Given the essentiality of the enzyme and that the reduction in gyrase level affected growth, altered levels of the enzyme should impact chromosome organization and other cellular characteristics. Thus, to understand the contribution of DNA gyrase to

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ACCEPTED MANUSCRIPT cell structure, several phenotypic characteristics of MsGyrCk were assessed. Upon gyrase depletion, the cells acquired rough and dry colony morphology (Figure 3A). Mycobacteria are known to form pellicles in standing cultures when grown in the absence of detergents [42]. When pellicle formation was assessed, in contrast to WT

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cells, the MsGyrCk was unable to form a visible pellicle (Figure 3A). To assess the

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biofilm-forming ability, cultures were grown in polystyrene plates and incubated at 30C.

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Unlike the WT strain, MsGyrCk failed to form a biofilm. Sliding motility is another

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surface-related phenotype of mycobacteria that can be assessed on soft agar plates. The gyrase-reduced strain appeared to be non-motile and did not show the typical halo

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formation, a characteristic of sliding motility (Figure 3A). The MsGyrCk strain also showed reduced ATP levels compared to that of the WT strain, indicating the impact on

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central metabolism (Supplementary Figure S4 A). Reduced gyrase levels affected the

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transcription of many genes (see later section). The qRT-PCR analysis shows that atpA, atpB, atpC genes are downregulated in the strain (Supplementary Figure S4 B).

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The effect of gyrase depletion on the cell morphology was also examined by

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scanning electron microscopy. The MsGyrCk cells were elongated, indicating a possible defect in cell division (Figure 3B). The frequency distribution plot showed a shift in the

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median length; the average size of the gyrase-depleted cells was 4 m, whereas the average size of WT cells is 2.5 m (Figure 3C). Since MsGyrCk cells were elongated, we investigated whether the essential cell division protein, ftsZ [43], was affected due to the supercoiling imbalance. A concomitant decrease in ftsZ level along with depletion of gyrase levels (Figure 4A, B) indicated a link between gyrase and cell division in M. smegmatis.

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ACCEPTED MANUSCRIPT To explore the effect of gyrase perturbation on nucleoid architecture, confocal microscopy was performed by DAPI staining of gyrase-depleted cells. Unlike the condensed nucleoid of the WT, the nucleoid of MsGyrCk appeared to be diffuse (Figure 4C). Reduction in gyrase level is known to alter the supercoiling status of the

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chromosome [2], [ 44]. To investigate the level of DNA supercoiling in the gyrase-

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depleted cells, reporter plasmid (pMV261) was isolated, and topoisomer distributions

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were analyzed by one-dimensional chloroquine gel electrophoresis. The depletion in gyrase level resulted in a significant reduction in supercoiling (Figure 4D,

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Supplementary Figure S5). The reduction of superhelical density in the plasmids from

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MsPRS and MsGyrCk indicated that reduction in gyrase level is likely to cause relaxation of the chromosome.

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encoding transcription machinery

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Perturbation of supercoiling alters expression of topology modulators and genes

The interplay between gyrase and topo I and the response on altering one’s

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activity on the other has been investigated in E. coli [1], [ 2], [ 45], [ 46], [ 47]. For

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example, topA mutants of E. coli grown in the presence of compensatory mutations in gyrA or gyrB that reduce the supercoiling activity of DNA gyrase [1], [ 2], [ 48].

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Similarly, when topo I expression in M. smegmatis was downregulated, it resulted in a decrease in gyrase levels [37], highlighting the importance of restoring topological balance in mycobacteria as well. Thus, one would predict that continuous suppression of gyrase levels should impact topo I expression. Indeed, the expression of the sole type I topoisomerase of M. smegmatis was reduced by 0.4-fold with gyrase repression in the knockdown strain (Figure 5A, B). Hence, a concurrent reduction in topo I

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ACCEPTED MANUSCRIPT expression appears to be a cellular strategy to bring about topological homeostasis. To investigate the impact of gyrase down-regulation on the expression of other topology modulators, transcript levels of two NAPs were assessed by qRT-PCR. Conditional knockdown of gyrase resulted in a 2.9-fold increase in hupB mRNA. Lsr2, a

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mycobacteria-specific NAP showed a 6-fold increase in mRNA when compared to WT

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cells (Figure 5B).

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Negative supercoiling facilitates transcription initiation. Gyrase is also required for the continuous removal of positive supercoils generated during transcription elongation.

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In turn, higher recruitment of topoisomerases to actively transcribed genes is observed,

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validating the model of twin supercoiled domains generated in vivo[18], [ 49]. Hence, to investigate the consequences of gyrase deprivation on transcription of actively

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transcribed genes, mRNA levels of sigA, sigB, 16srRNA and rpoB were measured.

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Expression of sigma factor sigA was reduced marginally, but sigB expression increased by ~10 fold (Figure 5B). Surprisingly, rpoB expression was increased 4-7-fold in

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MsGyrCk cells (Figure 5C). To examine whether there is an increased expression of

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RNAP, transcript levels of all the subunits were measured. Only rpoB and rpoC expression increased whereas expression of alpha (rpoA) and omega (rpoZ) subunits

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was mostly unaltered (Figure 5C). These results suggest that transcription of rpoB and rpoC is responsive to supercoiling. To examine the effect of altered supercoiling on ectopic gene expression, the transcript level of lacZ was measured using pMIND [32] based reporter plasmid where lacZ was cloned downstream of the tetracycline-inducible TetR promoter. The induction in the level of lacZ transcript was reported to be 4-fold lower in MsPRS strain compared

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ACCEPTED MANUSCRIPT to WT (Supplementary Figure S6). Similarly, when Rv3852 of M. tuberculosis which is not found in M. smegmatis [30] was expressed on inducible plasmid pJAM2 [31], its expression was reduced by 50% both at the transcript and protein level (Supplementary Figure S6 A, B). The altered transcription was further analyzed by

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ChIP using anti-RpoC antibody. The RNAP occupancy on the Rv3852 coding region in

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the gyrase depleted strain was 6-fold less than that found in the WT cells

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(Supplementary Figure S6 C). Taken together, the reduction in gyrase influenced the expression of genes in both chromosomal and plasmid contexts, implying that the

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overall transcription in the cell is affected due to a reduction in gyrase level.

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Gyrase reduction affects the RNAP occupancy over transcription units The difference in the levels of topoisomerases, NAPs, and transcription

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machinery indicated that gyrase depletion affected their transcription in the MsGyrCk

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strain. Impaired transcription could be due to changes in RNAP distribution over the transcription unit (TU) (Figure 5D). The relative RNAP occupancy in any given gene

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can be assessed by measuring its recruitment at promoter vs. open reading frame

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(ORF) [50]. The RNAP occupancy at the promoter and coding region of genes whose transcription was found altered in the MsGyrCk was determined by ChIP-RTPCR. The

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hupB and lsr2 ORFs showed significant enrichment of RNAP relative to their respective promoters, correlating with their increased transcription in the gyrase-depleted strain (Figure 5E). In contrast, the occupancy of RNAP at the topo I promoter was more than at the ORF, thus correlating with its reduced transcription in MsGyrCk. Notably, the RNAP recruitment both at the ftsZ promoter region and ORF was reduced in the

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ACCEPTED MANUSCRIPT MsGyrCk compared to that in the WT (Figure 5E). As a result, the expression levels are reduced upon gyrase reduction. Comparison of gyrase knockdown versus RST Earlier, the importance of DNA gyrase on cellular functions was investigated by

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treating the cells with gyrase inhibitors [14], [ 23]. When cells were treated with

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aminocoumarins (novobiocin and coumermycin), the cellular response included gyrase

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autoregulation [6], [ 7], [ 48]. Initial inhibition of gyrase resulted in induction of gyrase by RST. Thus, to compare the outcome of the two different approaches of perturbation of

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DNA gyrase (inhibitor treatment vs. knockdown) on gene expression, M. smegmatis

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cells were treated with 30 µg/ml novobiocin for different periods. An early time point (10 min) represents a condition in which gyrase is functionally inhibited (Figure 6 A, B),

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which will be referred to as pre-RST. However, upon longer exposure (30-90 min) gyrB

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and gyrA expression increased due to RST (Figure 6 B), confirming our earlier observations [7]. A similar pattern is seen with topA. Notably, ftsZ expression was

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severely affected immediately following novobiocin treatment. However, during RST, the

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ftsZ expression level was restored albeit at a later time than gyrase. These results reveal a close link between supercoiling and ftsZ transcription. However, the pattern of

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expression of the two NAPs is different. During the pre-RST condition, the expression of lsr2 remained close to that of the untreated levels but was elevated during RST. hupB showed a decrease in expression during pre-RST phase, and its levels were restored upon RST (Figure 6B).

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ACCEPTED MANUSCRIPT Reduction in gyrase renders M. smegmatis more susceptible to drugs Treatment of drug-sensitive TB involves a combination therapy having four firstline drugs [51], [ 52]. Further, fluoroquinolones are clinically validated second-line drugs for MDR strains of TB [53]. Hence, consequences of the reduction of intracellular gyrase

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on the MIC of drugs used for TB treatment was investigated. MsGyrCk cultures

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exhibited increased susceptibility to isoniazid (INH) and rifampicin [54], first-line anti-TB

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drugs. While 1-2 µg/ml of INH was able to bring about growth arrest in the MsGyrCk, the WT growth was inhibited at 4 µg/ml (Figure 7A). In the case of rifampicin, a 2-fold lower

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concentration led to growth inhibition of MsGyrCk compared to WT (Figure 7B).

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Treatment with moxifloxacin (MFX), a potent inhibitor of mycobacterial DNA gyrase [16], caused MsGyrCk growth inhibition at 0.156 µg/ml whereas a higher concentration (1.25

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µg/ml) was required to inhibit the WT growth (Figure 7C). Bedaquiline (BDQ), an ATP

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synthase inhibitor currently used for MDR TB treatment [55] led to the cessation of growth in the gyrase-depleted strain at 0.0312 µg/ml while the same drug concentration

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showed delayed growth of the WT (Figure 7D). Further, imipramine, a newly discovered

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mycobacterial topo I poison [56] delayed the growth of MsGyrCk at concentrations of 34 µg/ml and completely inhibited at 68 µg/ml compared to the 136 µg/ml dosage required

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for growth arrest of the WT cells (Figure 7E). The increase in the susceptibility of MsGyrCk strain could be due to an increase in drug uptake upon gyrase depletion. To validate this, an EtBr uptake assay was performed. The gyrase-depleted strain showed a 2-fold increased accumulation of EtBr than the WT suggesting increased permeability of the MsGyrCk (Supplementary Figure S7).

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ACCEPTED MANUSCRIPT DISCUSSION DNA gyrase is an essential component in all eubacteria, and hence it is no surprise that gyrase mutants of M. tuberculosis were not obtained by saturation transposon mutagenesis [57]. Earlier studies on the intracellular role of gyrase were

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mostly facilitated by E. coli ts mutants [1], [ 23], [ 24], [ 27], steady-state mutants [1], [ 2],

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[ 22] and use of gyrase inhibitors [6], [ 19], [ 21]. Instead of these conventional

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approaches, in the present experimental design, DNA gyrase was downregulated by rewiring the genetic switch. One of the advantages of using a repressible promoter

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system is the ability to study the function of essential genes by conditionally expressing

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them [28], [ 58], [ 59]. Furthermore, the inbuilt RST circuit of gyrase operon is circumvented by placing gyr operon under a promoter not subjected to autoregulation.

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By employing this gene silencing system, we show that manipulating the level of

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endogenous gyrase results in attenuation of transcriptional activity of many genes. Alteration in gyrase levels in M. smegmatis changed the DNA supercoiling leading to

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slower growth, severe phenotypic consequences such as altered colony morphology,

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lack of biofilms, reduced sliding motility and elongated cells. Gyrase depletion led to an alteration in expression of topo I and ftsZ, a major component of cell division. As a

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response to low gyrase levels, expression of NAPs, components of transcription machinery and chromosome architecture, were altered suggesting global changes in gene expression profile. The alterations in colony morphology, sliding motility and biofilm suggest an altered lipid composition of the bacterial surface in the MsGyrCk strain akin to the topo I cKD strain developed earlier [37] where a reduction in the transcripts of glycolipid synthesis and transport were seen.

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ACCEPTED MANUSCRIPT Being the sole supercoiling enzyme, it is apparent that gyrase is an essential component for cellular functions in bacteria. However, the minimum number of gyrase molecules needed for cell sustenance has not been estimated in any system so far. From our estimation, it appears that cell growth and sustenance is highly compromised

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when gyrase expression is reduced below 50% in M. smegmatis. Although the total

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number of gyrase molecules would vary between different bacteria, it is likely that a

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minimum threshold level needs to be maintained in any organism given the enzyme’s indispensable function. However, the actual minimum number of molecules could vary

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in diverse species owing to the differences in cellular characteristics and distribution of

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topoisomerases. An estimate of GyrA and GyrB in E. coli using immunogold staining revealed a range of 1000-3000 molecules per cell [60], but the minimum number

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required for cell growth is not known. The severity of the growth defect in the MsGyrCk

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could also be because gyrase is a key metabolic sensor and thus, its activity is also regulatable by cellular metabolic processes [61]. Gyrase activity depends on the ratio of

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ATP/ADP in the cell which in turn is a reflection of metabolic flux [62]. Rapidly growing

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cells have a higher ratio ATP to ADP compared to metabolically quiescent cells [63]. Changes in the ATP/ADP ratio affect gyrase activity in vivo thereby controlling

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supercoiling [12]. The MsGyrCk strain showed lower ATP levels compared to the WT strain which in turn may affect various pathways involving energy expenditure. With the decrease in gyrase levels, a decrease in topo I expression is observed in the MsGyrCk. Similarly, when topo I expression was reduced in M. tuberculosis and M. smegmatis [37], [ 64] gyrase expression was affected concomitantly. Given the pivotal role of gyrase ahead of transcription machinery to remove positive supercoils

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ACCEPTED MANUSCRIPT and the action of topo I on negative supercoils generated behind, both the enzymes have to be regulated to ensure topological balance. Alteration in the concentration of gyrase or perturbation in DNA supercoiling has different effects in different organisms, as illustrated previously in S. coelicolor [26], Salmonella and E. coli [65]. Notably, two

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clinical strains of Salmonella having mutations in gyrase genes exhibited diverse

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supercoiling and phenotypic properties, suggesting that variations in levels of

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topoisomerase activities can influence the physiology of the organism differentially [66]. Transcription of virulence genes is known to be modulated by DNA topology.

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In Salmonella, the expression of two type-III secretion systems involved in adaptation

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and pathogenesis inside the host showed sensitivity to chromosome supercoiling [67]. The observation that the virB gene promoter is inactivated in Shigella flexineri upon

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inhibition of DNA gyrase [68] indicated the importance of gyrase in the regulation of

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virulence. In Streptococcus pneumoniae, inhibition of DNA gyrase resulted in the activation of stress-responsive, virulent genes and simultaneous downregulation of

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several housekeeping genes involved in cell metabolism and growth [69]. Hence,

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understanding of topology control of gene expression exerted by M. tuberculosis gyrase is of certain importance.

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DNA gyrase is the master regulator of global DNA supercoiling which may potentially affect global gene expression. Hence, the depletion of both the essential topoisomerases with the downregulation of either of them would impact the DNA supercoiling. The increase seen in the expression of two topology modulatory proteinsHU and Lsr2 was unexpected. NAPs function as a buffer for topological perturbations, and therefore upregulation of their expression could be a response to altered DNA

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ACCEPTED MANUSCRIPT supercoiling to maintain topological homeostasis. Upregulation of rpoB and rpoC suggested the possibility of an overall increase in RNAP in response to supercoiling stress. However, rpoA and rpoZ expression remained largely unaltered suggesting that only the promoter for the former component (rpoBC) is supercoiling sensitive. Elevation

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in the level of the second major sigma factor, sigB, indicates that its promoter also

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responds to supercoiling perturbation. Being involved in a variety of stress response,

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the sigB levels need to be regulated; the promoter of sigB appears to respond to supercoiling. A similar observation was reported in E. coli wherein a template with low

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superhelical density favored binding of stationary phase-specific σ38 to the target gene

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while the binding of housekeeping σ70 was optimal with the negative supercoiled DNA [70].

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During E.coli DNA replication, gyrase-mediated negative supercoils facilitate both

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the unwinding and the topo IV-dependent unlinking of parental strands [71]. Although, the involvement of both DNA gyrase and topo IV in chromosome partitioning during cell

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division was implicated before topo IV was discovered [72], now it is apparent that the

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latter enzyme is the principle decatenase [4]. Since mycobacteria lack topo IV, DNA gyrase doubles up as a decatenase [16], [ 17], [ 73] indicating that the enzyme might

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have a major role in chromosome segregation and cell division. The observation by Pang et al. that the GyrB652 ts mutant having active gyrase protein was prone to DNA replication collapse near the terminus under non-permissive temperatures indicated the enzyme’s role in replication [74]. MsGyrCk showed a temperature-sensitive and elongated phenotype hinting towards the possibility of abnormal replication. Another factor that could lead to an elongated phenotype is abnormal segregation after

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ACCEPTED MANUSCRIPT replication. Further study will be required to address these aspects. Upon gyrase depletion, a concomitant reduction in ftsZ levels was observed establishing a closer connection between gyrase and cell division in mycobacteria. Chromosome segregation and cytokinesis are functionally linked [75]. Reduced ftsZ levels in M. smegmatis

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resulted in increased cell length and reduced cell viability [43]. The role of topo I in cell

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division and segregation has also been reported in E. coli where topA null mutants

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showed supercoiling-independent segregation defects [76]. As seen, reduced gyrase levels slow down cell growth and division when coupled with the lower expression of

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ftsZ. Thus, the reduced levels of the gyrase and topo I might have both a direct and

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indirect role in the cell division process of mycobacteria.

The reduction in DNA gyrase influenced the expression of genes in both

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chromosomal and plasmid context implying that the overall transcription in the cell is

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affected due to the altered supercoiling environment. The recruitment of both DNA gyrase and topo I to active TU’s in mycobacteria validated the twin supercoiling

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domains generated in vivo [18], [ 49]. The decrease in the levels of both the

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topoisomerases described above should impede the transcription process given the close coupling of topology and transcription. A high polymerase density at the promoter

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region relative to the gene body is indicative of poised RNAP near the TSS [50]. Under circumstances of rapid transition from initiation to elongation, the RNAP levels at the promoter and in the corresponding coding sequence will be equivalent. However, if the transition is slow, the association of RNAP would be more at the promoter than in the coding region. Since gyrase participates ahead of the transcription machinery to remove the torsional strain, the alteration in gyrase level can affect the distribution of RNAP

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ACCEPTED MANUSCRIPT across the TU’s as seen in the case of downregulated genes. Although gyrase depletion resulted in severe growth defect and impaired transcription of several genes, RNAP levels as such remain unaffected except the increased expression of the rpoBC operon. Hence, the decreased transcription of the genes is likely due to reduced RNAP

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recruitment or the effect on subsequent steps of transcription. Since the DNA in

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MsGyrCk is in a relatively relaxed state with both gyrase and topo I being

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downregulated, the genes that are found upregulated have increased transcription initiation (reduced RNAP occupancy at promoter) leading to higher RNAP density at the

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ORF. It has been suggested that a battery of RNAPs tandemly located in the ORF

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annihilate the positive and negative supercoils generated during transcription elongation [77]. In such a situation, multiple transcription elongation complexes would overcome

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the supercoil barriers. Indeed, this phenomenon is observed during rRNA transcription

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[78].

The increased susceptibility of MsGyrCk to the drugs tested appears to be a

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consequence of reduced gyrase levels. Addition of ATc lowers gyrase level similar to a

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situation of gyrase activity inhibition, and therefore the efficiency of novel molecules for triggering cell death can be assessed. The lowering of MIC for moxifloxacin and

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imipramine was unanticipated since their mode of action involves arrest of topoisomerase reaction after first transesterification step leading to DNA breaks. Lowering of gyrase levels reduce cell growth, replication, and global transcription. Replication and

transcription

generate supercoils and

recruit topoisomerases

throughout the genome. Upon reduction of transcriptional activity, sites for DNA gyrase and topo I recruitment on chromosome decreases [18], consequently compromising

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ACCEPTED MANUSCRIPT moxifloxacin and imipramine-induced DNA cleavage by DNA gyrase and topo I respectively. A decrease in MIC for the drugs tested indicates a common mechanism of increased drug sensitivity when the levels of gyrase are lowered and can be used for screening new anti-tubercular drugs which may act synergistically with gyrase inhibitors.

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Acknowledgments

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We thank K. Drlica for critical inputs and editing of the manuscript, S. Bhowmick for

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proofreading of the manuscript, R. Manganelli for plasmid construct pFRA50 and pFRA42B, P. Ajitkumar for MsmftsZ antibody and members of VN laboratory for

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valuable suggestions.

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Funding

The work in VN laboratory is supported by the grant from Department of Biotechnology,

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Government of India(MCB/VNR/DBT/496) and by the UKIERI grant from British council

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(DST-2012-2016/091) and Indian Institute of Science. VN is a J.C. Bose Fellow of Department of Science and Technology, Government of India.

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Conflict of interest statement: None declared

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Figure 1. Construction of M. smegmatis DNA gyrase conditional knockdown strain.

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A) Strategy used to replace native promoter (Pgyr) of M. smegmatis gyrB with Pptr by single-site crossover. The 966 bp of the gyrB-NTD coding region was cloned in pFRA50

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to obtain plasmid pFRA50-MsGyrB which was then electroporated in M. smegmatis cells harboring pFRA42B. B) Immunoblot was performed with cell lysate of WT,

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promoter-replaced strain (MsPRS) and MsGyrCk strain treated with 200 ng/ml anhydrotetracycline (ATc) to check reduction of gyrase expression. Ponceau-S staining of blot served as loading control. Bar graphs show the band intensities of MsPRS and MsGyrCk + ATc relative to the WT band intensity. Values are represented as mean ± SEM and n=3. ***p<0.001 versus WT.

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Figure 2. Importance of gyrase for growth and cell sustenance. A) Effect of gyrase depletion on the growth of WT and MsGyrCk at 37°C and 30°C in

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Middlebrook 7H9 broth. B) Effect of increasing concentrations of ATc on the growth of MsGyrCk. C) Growth under constant gyrase repression was monitored by supplying

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ATc after every 12hr intervals in media. Sterile medium and untreated culture were used as controls for all growth experiments. D) Growth of WT and MsGyrCk on Middlebrook

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7H10 agar supplemented with 200 ng/ml ATc in serial dilutions of culture. E) Quantitative western analysis. Different concentrations of purified GyrA and 200 µg of total cell lysate from WT and MsGyrCk cells treated with 500 ng/ml ATc were resolved on 8% SDS-PAGE and immunoblotted with the anti-GyrA antibody. Graph of immunostaining intensity (arbitrary units) versus relative protein concentration (ng) shows a linear relationship between these two parameters for range the of protein concentration chosen.

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Figure 3. Reduction of gyrase level alters the phenotype of MsGyrCk.

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A) Colony morphology was observed by growing cells on 7H10 agar plates for 9 days. Biofilm and pellicle formation was measured in static cultures of 7H9 broth without

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Tween-80. The gyrase-depleted strain was severely compromised in forming any biofilm or pellicle. Sliding motility was determined on 0.4% agar wherein the WT was seen to

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form a halo of motile cells from the central point, but the MsGyrCk was non-motile and unable to slide on soft agar. B) Scanning electron microscopy of WT and MsGyrCk grown in the presence of 200 ng/ml ATc for 6 hr. MsGyrCk cells appeared elongated compared to WT cells. C) Frequency distribution of the cell size (n = 300) in WT and MsGyrCk strain.

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Figure 4. Reduction in gyrase level causes genome decompaction. A) The relative level of ftsZ mRNA was determined in MsGyrCk with respect to that in

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WT by qRT-PCR and B) immunoblot using anti-MsmftsZ antibody. ‘+’ indicates culture treated with 100 ng/ml ATc and ++ indicates 200 ng/ml ATc. C) The nucleoid of the

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conditional mutant was visualized by DAPI staining (showed as a pseudo-colored image) by fluorescence microscopy under 100x objective. D) DNA supercoiling changes of pMV261 isolated from the WT, MsPRS and the MsGyrCk after exposing it with 200 ng/ml ATc for 6 hr. Topoisomer distributions were obtained from the electrophoretic mobility of pMV261 in agarose gel containing 2.5 µg/ml chloroquine. Migration is from top to bottom. Lane 1 shows negatively supercoiled topoisomers, lanes 2 and 3 are positively supercoiled. Also see supplementary figure S5 depicting the handedness of the topoisomers in no CQ, 1.5 µg/ml, 2.5 µg/ml and 10 µg/ml CQ conditions. OC, open circles; N, nicked; CQ, chloroquine. 31

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Figure 5. The influence of reduced gyrase level on the expression of genes.

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A) Intracellular levels of DNA gyrase and topo I from cell lysate of WT and MsGyrCk

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detected by immunoblot analysis. *, #p<0.05 versus MsGyrCk. Ponceau-S staining of blot served as loading control. B) mRNA levels of various topology modulators and

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house-keeping genes determined by absolute quantification of each gene and represented as fold-change. *p<0.05, ***p<0.001 versus WT+ ATc. C) Measurement of

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transcripts of the RNAP core enzyme subunits by qRT-PCR. Means and error bars (representing SD) from three separate experiments are shown. *p<0.05, **p<0.01

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versus WT+ ATc. D) Schematic showing correlation of RNAP (shown in pink) occupancy over promoter and ORF with transcription. More RNAP at ORF suggests increased transcription while the reverse condition with a higher density of RNAP at promoter than ORF leads to less transcription. E) The occupancy of RNAP over promoter and coding sequence was determined by ChIP using anti-RpoC antibody followed by detection of specific regions by qRT-PCR. *p<0.05, ***p<0.001 versus promoter.

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Figure 6. Expression upon novobiocin treatment.

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A) Immunoblot detection of GyrA levels to establish the pre-RST and RST phase upon treatment with 30 µg/ml novobiocin for different time points. *p<0.05, **p<0.01versus 0

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min. Ponceau-S staining of blot served as loading control. B) RT-qPCR analysis to detect expressions of gyrB, gyrA, topA, hupB, lsr2, ftsZ and rpoB upon novobiocin

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treatment. RNA was isolated after exposing cells with 30 µg/ml novobiocin for time points indicated. The data is normalized with 16s rRNA, and error bars represent values

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from 3 independent experiments.

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Figure 7. Increased drug susceptibility of MsGyrCk. WT and gyrase conditional-knockdown strain were grown in the presence of various concentrations (0 to 8 μg/ml) of isoniazid (A), (0 to 256 μg/ml) rifampicin (B), (0 to 1.25 μg/ml) moxifloxacin (C), (0 to 4 μg/ml) bedaquiline (D) and (0 to 136 μg/ml) imipramine (E). The growth was followed over a period of 96 hr with O.D being measured every 3 hr. The untreated cultures were used as controls. 34

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Supplementary Data:

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Figure S1. Validation of gyrase conditional knockdown strain in M. smegmatis. Promoter replacement was confirmed by PCR with genomic DNA isolated from

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MsGyrCk (lane 2), MsPRS (lane 3) and WT (lane 4).

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Figure S2. ATc concentration-dependent repression of MsGyrCk growth. WT, MsPRS and MsGyrCk were grown in Middlebrook 7H9 broth containing 100 ng/ml-

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600 ng/ml ATc. Growth was measured every 3 hr for 4 days. 1 µl of ATc treated cultures

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was spotted onto solid medium without ATc to check cell survival.

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Figure S3: Estimation of minimal gyrase required for cell sustenance.

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Representative image showing viable colonies obtained when WT and MsGyrCk cells were treated with 500 ng ATc and then plated on 7H10 agar without ATc. To estimate

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the minimum number of gyrase molecules, CFU/ml was calculated and correlated with

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the amount of gyrase expressed per cell.

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Figure S4. Quantification of ATP levels.

A) Exponentially growing cultures of WT and MsGyrCk were incubated with

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recombinant firefly luciferase. The light emitted due to luminescence is directly proportional to the ATP molecules in the cell. B) mRNA level of ATP synthase genes

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quantified by qRT-PCR.

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Figure S5. DNA supercoiling changes of the reporter plasmid isolated from WT,

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MsPRS, MsGyrCk treated with 200 ng/ml ATc. The electrophoretic mobility of pMV261 topoisomers in agarose gel with (1.5 µg/ml, 2.5 µg/ml, 10 µg/ml) and without

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CQ. In absence of CQ, SC DNA migrates faster than relaxed and linearized DNA. With

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increasing concentrations of CQ, a decrease in negative supercoils was observed in WT while positive supercoils were introduced in plasmid from PRS and cKD. Migration is from top to bottom. L, linearized plasmid; OC, Open circles; SC, supercoiled; CQ,

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chloroquine.

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Figure S6. Influence of reduced gyrase level of ectopically expressed genes A) Western blot detection of Rv3852 level in WT and MsPRS before (-) and after (+)

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induction with 2% acetamide. Ponceau-S staining of blot served as loading control. B) Transcription of the Rv3852 gene in the WT and MsPRS cells. C) Occupancy of RNAP

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on Rv3852 ORF was quantified by ChIP RT-PCR. D) mRNA level of lacZ in pMIND

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vector expressed in WT and MsPRS strains upon induction with tetracycline.

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Figure S7. Cell wall permeability of the MsGyrCk strain.

Exponential phase cultures of WT and MsGyrCk were treated with 0.5 μg/ml EtBr.

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Uptake of EtBr by gyrase-depleted strain was more compared to the WT strain after 10

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min incubation.

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ACCEPTED MANUSCRIPT References:

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ACCEPTED MANUSCRIPT Highlights: 1. DNA gyrase levels are restored by relaxation stimulated transcription. 2. Replacing native Pgyr promoter with supercoiling-insensitive promoter ensures continuous gyrase repression.

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3. A minimum threshold of gyrase is required for the organism’s survival.

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4. Gyrase depletion alters cell morphology, growth and drug susceptibility.

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5. Reduced gyrase level alters RNA polymerase occupancy and transcription.

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