Microbiology Papers in Press. Published December 16, 2014 as doi:10.1099/mic.0.000014

Microbiology Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis --Manuscript Draft-Manuscript Number:

MIC-D-14-00090R1

Full Title:

Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis

Short Title:

Perturbation of topoisomerase I expression in M. smegmatis

Article Type:

Standard

Section/Category:

Cell and Molecular Biology of Microbes

Corresponding Author:

Valakunja Nagaraja Indian Institute of Science Bangalore, Ka INDIA

First Author:

Wareed Ahmed

Order of Authors:

Wareed Ahmed Shruti Menon Pullela VDNB Karthik Valakunja Nagaraja

Abstract:

The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution is varied in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single Type I (TopoI) and a single Type II (Gyrase) enzyme, Mycobacterium. smegmatis (Msm) and other members harbor additional relaxases. TopoI is essential for Mtb survival. However, the essentiality of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extreme slow growth and drastic change in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins resulting in a diffused genome architecture. The proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. The RNA polymerase (RNAP) distribution on the transcription units was found to be affected in the TopoI-depleted cells suggesting the global alteration in the transcription. The study thus highlights the essentiality of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. Decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation causing severe downstream effects.

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Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis Wareed Ahmed1, Shruti Menon1, Pullela VDNB Karthik1 and Valakunja Nagaraja1, 2

1

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 2

Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

*Correspondence: Prof. V. Nagaraja E-mail: [email protected] Tel.No. +91-0802360 0668 Fax No. +91-080-2360 2697 Content category: Cell and Molecular Biology of Microbes Running title: Perturbation of topoisomerase I expression in M. smegmatis Key words: Mycobacteria, Topoisomerase, Gene expression, Essential gene, Cell phenotype, transcription Word count: Summary: 241 Main Text: 6242 Figures: 8

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SUMMARY The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution is varied in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single Type I (TopoI) and a single Type II (Gyrase) enzyme, Mycobacterium. smegmatis (Msm) and other members harbor additional relaxases. TopoI is essential for Mtb survival. However, the essentiality of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extreme slow growth and drastic change in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins resulting in a diffused nucleoid architecture. The proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. The RNA polymerase (RNAP) distribution on the transcription units was found to be affected in the TopoI-depleted cells suggesting the global alteration in the transcription. The study thus highlights the essentiality of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. Decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation causing severe downstream effects.

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INTRODUCTION The genome compaction is necessary to accommodate it within the small intracellular compartment of a bacterial cell. Bacterial DNA compaction is facilitated by the introduction of negative supercoiling activity of DNA topoisomerases (Deng et al., 2005; Saier, 2008; Wang, 2002). Negative supercoiling enhances the free energy of the DNA which facilitates the melting of the promoter elements during transcription initiation. Potentially, the alteration in the genome supercoiling/DNA topology can affect the transcription (Travers & Muskhelishvili, 2005). Molecular and genetic evidences suggest that several environmental cues modulate the supercoiling/DNA topology of the bacterial genome (Drlica, 1992) which consequently influences the gene expression (Dorman, 1991). Anaerobic growth, osmolarity and temperature have been shown to affect the supercoiling of the DNA (Dorman et al., 1988; Goldstein & Drlica, 1984; Higgins et al., 1988). The genome supercoiling, in turn appears to sense, interpret and co-ordinate the expression of vast sets of genes contributing to the adaptation to various environments (Dorman, 1991; Dorman, 2006). The supercoiling of the bacterial genome is maintained by the countervailing activities of the DNA gyrase and TopoI (DiNardo et al., 1982; Pruss et al., 1982; Wang, 1985). TopoI relaxes excess negative supercoiling accumulated due to the action of replication and transcription machineries while DNA gyrase introduces negative supercoiling in an ATP-dependent manner to maintain optimal negative supercoiling (Champoux, 2001). The metabolic state or the environmental cues alter the [ATP/ADP] ratio in the cell (Hsieh et al., 1991; Westerhoff et al., 1988) which in turn would influence the DNA gyrase activity resulting in change in the superhelical density of the genome (Drlica, 1992; Hsieh et al., 1991). Any alteration or imbalance in topoisomerase expression and/ or activity inside the cell can modulate the gene expression. 3

Indeed, the mutations in the genes or the actions of inhibitors on these major topology modulatory enzymes lead to various pleotropic effects on the cellular processes of various bacteria. For instance, in a TopoI-deficient mutant of Salmonella Typhimurium, osmotic induction of invA gene is affected leading to diminished invasiveness (Galan & Curtiss, 1990). The transcription of the invasive genes of the organism is also perturbed by the inhibition of DNA gyrase (Dorman & Ni Bhriain, 1993). In Escherichia coli, Streptococcus pneumonia and Haemophilus influenza total transcriptome was altered upon DNA gyrase inhibition suggesting the global regulatory role of the genome supercoiling (Ferrandiz et al., 2010; Gmuender et al., 2001; Peter et al., 2004). Further, proteomic study with the TopoI and DNA gyrase mutant revealed an altered proteome of E. coli (Steck et al., 1993). From these studies, it is evident that DNA topoisomerases play a crucial role in the physiology and gene expression of eubacteria (Hatfield & Benham, 2002). The genus Mycobacterium comprises a variety of organisms including various slow growing pathogens such as M. tuberculosis, M. leprae, M. marinum, M. abscessus as well as the well-studied non-pathogenic fast growing M. smegmatis. In contrast to the slow growing Mtb which contains a single TopoI for genome relaxation, Msm has an unusual type II enzyme, TopoNM and an uncharacterized type IB topoisomerase apart from TopoI to carry out DNA relaxation function (Jain & Nagaraja, 2005). While the indispensability of TopoI in Mtb has been demonstrated recently (Ahmed et al., 2014), the presence of surplus DNA relaxation enzymes raises the question about the importance of TopoI in Msm for growth and gene regulation. Having additional DNA relaxation enzymes in the Msm genome would suggest the dispensability of TopoI as the other two enzymes could cope up with the cellular DNA relaxation burden. Further, the role of topoisomerases and genome supercoiling in the physiology of mycobacteria 4

is yet to be explored. Several studies have unraveled the distinctive features of mycobacterial TopoI, such as site-specific DNA binding, high processivity and ability to bind both the singleas well as double-stranded DNA (Bhaduri et al., 1998a; Bhaduri et al., 1998b). These distinct properties of Msm TopoI may contribute for vital functions and regulatory roles. Thus, to monitor the influence of the alteration of TopoI level on mycobacterial physiology and growth, conditional knock down strain of TopoI is generated in Msm using the TetR-pip based system (Boldrin et al., 2010). The mutant with reduced expression of TopoI exhibited severe growth defect, altered nucleoid architecture and phenotypic variations indicating the global regulatory role of TopoI in the biology of Msm. METHODS Bacterial strains, growth media and transformation conditions The following bacterial strains were used: E. coli DH10B (laboratory stock), M. smegmatis mc2155 (laboratory stock). E. coli strains were grown at 37°C in Luria–Bertani (LB) broth or on LB agar plates. Mycobacterial strains were grown at 37°C in Middlebrook 7H9-broth (Difco) or on 7H10-agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80. For growth of MsPptrtopoI (topoI conditional mutant), the medium was supplemented with 10% ADC (Albumin, Dextrose, and NaCl) and cultures were grown at 30°C. Antibiotics were added to the media, at the following concentrations: Streptomycin (Sm): 20 µg ml-1; Kanamycin (Km): 50 µg ml-1; Hygromycin (Hyg): 150 µg ml-1 (E. coli) or 50 µg ml-1 (Msm). Anhydrotetracycline (ATc) (Sigma-Aldrich) was added when required, at final concentrations from 10 ng ml-1 to 200 ng ml-1.

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Construction of conditional mutant of Topoisomerase I (TopoI) in M. smegmatis The first 750 bp fragment of Msm topoI was cloned downstream of the ptr promoter region in the suicide plasmid pFRA50 to obtain pFRA50-MstopoI. In order to replace the promoters of topoI with the Pip controlled promoter Pptr, Msm cells were electroporated with 2 µg of plasmid (pFRA50-MstopoI). Recombinant colonies were selected on 7H10-agar plates, containing Hyg (50 µg ml-1). Integration of the plasmid via insertional duplication was confirmed by PCR. The resulting recombinant strain was transformed with the integrative plasmid pFRA42B (containing the TetR/Pip system) to obtain Msm topoI conditional mutant (MsPptrtopoI). For complementation analysis, TopoI over expression constructs were generated in the pMIND vector system (Blokpoel et al., 2005). The MstopoI gene was amplified from pPVN123 (Jain & Nagaraja, 2006). The PCR products were digested with NdeI and EcoRV and cloned into the NdeI and EcoRV linearized pMIND vector. Clones were confirmed by double digestion with NdeI and BamHI digestion and the expression of MstopoI in Msm cells was monitored by immuno-blotting. Growth analysis of MsPptrtopoI The colonies of MsPptrtopoI were grown on 7H10-agar at 30°C. The cultures of the conditional mutant were grown at 30°C in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.05% Tween-80 and 10% ADC. Wherever needed, MsPptrtopoI and WT cultures were treated with 10 ng ml-1to 200 ng ml-1 ATc for indicated period of time. For measuring the growth of Msm and mutant, exponential phase cultures (A595 nm = 0.4-0.6) were taken and diluted in 7H9-media to A595 nm = 0.02. Growth curve profiles were obtained using the BioScreen growth curve analyzer and the growth curve plots were generated using GraphPad Prism (version 5.0).

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Immunoblot analysis Proteins were separated on 8% SDS PAGE and transferred to PVDF membranes. Membranes were incubated in PBS blocking buffer (10 mM sodium phosphate, pH 7.5, 150 mM NaCl, 0.05% Tween 20) with 2% (w/v) BSA for 2 h prior to incubation with primary antibodies diluted (1:20,000) in PBS with 2% BSA for 2 h. Membranes were washed in PBST (0.2% Tween 20) three times, and incubated with Horseradish peroxidase (HRP) conjugated goat polyclonal antimouse IgG or goat polyclonal anti-rabbit IgG secondary antibodies (GE Amersham) (dilution 1: 20000) for 2 h followed by washing three times with PBST. Protein bands were visualized by chemiluminescent substrates (Millipore). Scanning Electron Microscopy (SEM) and Fluorescence (DAPI) Microscopy 5-ml culture aliquots were concentrated by centrifugation (5000xg) before suspending in fresh Middlebrook 7H9 medium. Aliquots of concentrated cells (at densities of 5 x 108 CFU ml-1) were placed on poly-L lysine-coated coverslips in 24-well tissue culture plates. Bacteria were allowed to settle for 30 min before gently decanting and adding 1 ml solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer (pH 7.4) with 0.2 M sucrose. Samples were treated with 2% OsO4 in 0.1 M sodium-cacodylate buffer for 2 h at room temperature. A series of sequential ethanol dehydrations were performed for 10 min each (30%, 50%, 70%, 95%, and 100%) before drying the samples under vacuum desiccators. Samples were gold sputter coated, and imaged with a SEM Quanta 200 scanning electron microscope. For fluorescence microscopy, mycobacterial cells were fixed by exposure to toluene (2%) and Triton X-100 (1%) at 4°C overnight. Prior to staining of the DNA, the Msm cells were washed, resuspended in phosphate buffered saline (PBS; 10 mM sodium phosphate, pH 7.4, 150 7

mM NaCl) and treated with lysozyme (2 µg ml-1). The cells were stained with DAPI (0.5 µg ml1

) for 15 min at room temperature and the samples were examined under a confocal microscope

(Zeiss) equipped with a 100x objective. Plasmid isolation and chloroquine/agarose gel electrophoresis Exponential phase cultures of WT and mutant (MsPptrtopoI) harboring the pMV261 plasmid were treated with glycine (1% w/v) for 3 h followed by harvesting of the cells. The plasmids were isolated using the QIAGEN Plasmid Midi Kit as per the manufacturer’s instructions. 2 µg of the purified plasmids were subjected to choloroquine/agarose gel (1.2 % w/v) electrophoresis using 1X TAE buffer containing 2.5 µg ml-1 chloroquine di phosphate (Sigma Aldrich, USA). Electrophoresis was carried out at room temperature by applying a voltage gradient of 2V cm -1 for 16 h. Following the electrophoresis, the gel was washed with double distilled water and topoisomers were visualized by staining with EtBr (0.5 µg ml-1). Biofilm, Pellicle and motility assays Biofilm formation was assessed as described previously (Ghosh et al., 2013; Recht et al., 2000). 100 µl of 7H9 medium supplemented with 0.5% Casamino acids (Casein Hydrolysate) was used per well in a 96-well PVC microtiter dish. The medium was inoculated with the exponential phase cells and microtiter dishes were incubated at room temperature for 48 h and 96 h for the WT and the mutant respectively (till the A595 nm = 0.5). The wells were rinsed twice with water, and 120 µl of a 1% cell-staining solution of crystal violet (CV) was added. Plates were incubated at room temperature for 30 min, rinsed with water three times, and scored for CV staining. Quantitation of biofilm formation was performed by extracting the biofilm-associated CV with 100% ethanol for 1 h and measuring the optical density at 570 nm. Pellicle formation was

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monitored by growing standing cultures of mycobacteria in Middlebrook 7H9 medium without Tween-80 at 37°C. Motility assays were carried out as described previously (Martinez et al., 1999). Briefly, cells were cultured in 7H9 medium to mid-logarithmic phase (A595

nm

= 0.4 to 0.6) before

spotting 2 µl aliquots onto motility medium consisting of 7H9 supplemented with 0.5% Casamino acids, 0.2% glycerol, solidified with low melting point agarose (wt/vol 0.4%). The inoculated plates were incubated for 24 h to 96 h at 30°C in plastic bags containing moistened tissue paper to ensure that the bacteria grew under humidified conditions. Purification and analysis of Glycopeptidolipids (GPLs) The GPLs from Msm cells were purified as described earlier (Khoo et al., 1996). Briefly, lipids were extracted by treating 5 g (dry weight) cells at exponential phase with chloroform/ methanol (2:1) for 24 h at room temperature. The organic supernatant was dried and re-dissolved in chloroform/methanol (2:1) and treated with an equal volume of 2 M NaOH in methanol at 37 °C for 30 min then neutralized with a few drops of glacial acetic acid. After drying, the solvent lipids were dissolved in chloroform: methanol: water (2:1:1). The aqueous layer was discarded and the organic layer containing the lipids was concentrated. The de-acylated lipids were spotted on to a silica-coated TLC plate (Merck) and mobilized with chloroform/methanol (9: 1). The sugar-containing lipids were visualized by spraying the plate with 10% H2SO4 in 1% 1-Naphthol (in ethanol) followed by heating at 120 °C for 10 min.

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RNA extraction and qPCR RNA was extracted from Msm cells using a Qiagen RNeasy kit according to the manufacturer's protocol. From the total RNA, cDNAs were synthesized using High capacity cDNA reverse transcription kit (Applied Biosystems). cDNA generated with random primers was used for quantitative real-time PCR (qPCR), with SYBR green as the indicator dye. The expression of the genes was quantified after normalization of RNA levels to the expression of the sigA gene. The qPCR cycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 57°C for 30 s, and 72°C for 20 s. EtBr uptake assay The exponentially grown cells were diluted to A595 nm= 0.4 and treated with the EtBr (0.5 µg ml1

). The EtBr loaded cells were centrifuged at 4000 rpm for 3 min and resuspended in EtBr-free

PBS containing 0.4% glucose. After adjusting the O.D.595

nm

to 0.4, aliquots of 100 µl were

transferred to a 1.4 ml cuvette. Fluorescence was measured in the fluorimeter (Hitachi) using the 290 nm band-pass and the 580 nm high-pass filter as the excitation and detection wavelengths respectively. Two dimensional gel electrophoresis and Mass spectrometry (2D-MS) Msm (WT) and MsPptrtopoI cells were grown in 7H9 medium at 30°C to an A595 nm = 0.6. The cells were harvested, washed twice with PBS and resuspended in lysis buffer (10 mM HEPES, pH 8.0, 7% urea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 2 M thiourea, 10 mM dithiothreitol, followed by sonication. The lysate was processed with a ReadyPrep 2-D Cleanup Kit (Bio-Rad). Immobilized pH gradient strips, pH 3 to 10 (Bio-Rad; 17 cm), were rehydrated with 300 μg of total protein lysate. Isoelectric focusing (IEF) was carried out on a Protean i12 IEF Cell (Bio-Rad) using the following protocol: (i) 0 to 500 V linear for 2 10

h, (ii) 250 V rapid for 2 h, (iii) 250 to 6,000 V linear for 3 h, and (iv) 6,000 V constant to 50 kVh. After equilibration, the strips were loaded and resolved on 12% SDS-PAGE. The gels were stained with a ProteoSilver Kit (Sigma-Aldrich), and the spots which differed in intensity between the samples were identified by mass spectrometry. Chromatin immunoprecipitation (ChIP) and RT PCR ChIP with exponentially grown Msm cultures was carried out as described previously (Uplekar et al., 2012). Briefly, formaldehyde cross-linked cells were sonicated to shear the DNA by Bioruptor (Diagenode). The fragmented DNA was immune-precipitated by using the anti-RpoB antibody and purified. The resulting ChIP-DNA was subjected to Real time PCR analysis to determine the enrichment of the target DNA in the IP sample over the mock-IP sample. The results were expressed as the enrichment (ER) of RNAP on target DNA in the mutant normalized to the enrichment value of the WT samples. RESULTS Growth analysis of the conditional knock-down strain The topoI knock down strain of Msm was generated by replacing the native promoter of topoI with ptr promoter as described in Methods. The successful replacement of the promoter was confirmed by PCR analysis (data not shown) and the level of TopoI in the knock-down strain was monitored by immunoblotting. Notably, the expression of TopoI in the mutant strain was found to be 1.5 fold lower than the WT strain, even in the absence of anhydrotetracycline (ATc) (Fig.1a). Addition of ATc led to further 2.5 fold reductions in the level of the protein. Thus, MsPptrtopoI strain itself is a knock down strain were the TopoI level was intrinsically lower and the level could be further reduced by the addition of ATc.

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Analysis of the growth indicated that TopoI mutant cells took 8 days to appear as colonies on the 7H10-agar compared to the WT where the colonies formed in 4 days. The slower growth of the mutant could be attributed to the reduced level of TopoI (Fig. 1a). Notably, the mutant failed to grow in the Middlebrook 7H9 medium used for the growth of WT cells and the growth was resumed only upon supplementation of 10% ADC in the medium (Fig. 1b). Additionally, the cultures showed temperature sensitivity and exhibited very slow growth at 37 °C; mutant cultures reached the exponential phase after 96 h (data not shown). The growth was improved by lowering the temperature to 30°C. All the cultures were therefore grown in the presence of 10% ADC at 30°C. From these observations, it is apparent that an optimal TopoI expression is required for normal growth of Msm. To further evaluate the necessity of TopoI for mycobacterial growth, the cultures were grown in the presence of different concentrations of ATc (Fig. 1c). The analysis indicated a prolonged lag phase of the TopoI depleted cells compared to the WT. With an increase in the ATc concentrations, the growth was severely compromised. Importantly, the growth of the mutant was rescued upon complementation with the plasmid copy of Mtb topoI (MttopoI) or Msm topoI (MstopoI) (Fig. 1d) confirming that the reduced TopoI activity inside the cell resulted in reduced growth. The rescue of Msm cells depleted of TopoI by complementing with MttopoI expressing plasmid suggests the similar in vivo roles of the enzymes, in accordance with their similar biochemical properties (Godbole et al., 2012). The requirement of additional growth supplements for the growth of the mutant indicated that the cellular metabolism is affected in the strain. Colony morphology and surface phenotypes of the mutant The severely compromised growth rate, requirement of nutrient supplements and low temperature for the growth of TopoI mutant suggested an altered metabolism which could affect 12

colony and cell morphology. Indeed, the TopoI depleted strain acquired rough colony morphology and the cells tended to cluster towards the center of the colony while the WT strain maintained the smooth colony morphology (Fig. 2a). Further, the SEM analysis of the cells of the conditional mutant strain revealed an irregular cell structure and bulb-like protrusions on the cell surface indicating a defective cell surface (data not shown). Change in colony morphology and cell surface of the mutant cells suggested the modification of other cell surface related phenotypes. Because of high lipid content, mycobacteria grown in the absence of detergents form a pellicle in the standing cultures (Etienne et al., 2002). In contrast to the WT strain, the mutant was unable to form a pellicle (Fig. 2d). The pellicle is the biofilm formed at the air-water interface (Branda et al., 2005; Solano et al., 2002) and a co-relation exists between the ability to form pellicle and biofilm. To better assess and quantify the potential of the mutant to form a biofilm, cultures were grown in polystyrene plates. Biofilm forming ability of the mutant was significantly compromised (~ 6 fold) as compared to the WT (Fig. 2c). Another surface related phenotype in Msm is the sliding motility, which is restricted to smooth colonies (Agusti et al., 2008). Since, the mutant acquires rough and dried colony morphology, the sliding motility was evaluated. The mutant was drastically affected in the sliding motility and did not show the typical halo formation, a characteristic of the sliding motility (Fig. 2b). Notably, the surface phenotypes were rescued by the ectopic expression of MstopoI (Fig. 2). All these results indicate that the reduction of TopoI level in Msm confers pleotropic effects resulting in altered surface properties and cell phenotypes of the mutant strain.

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Lipid profile of TopoI-depleted Msm cells Colony morphology, sliding motility, pellicle and biofilm formation are influenced by the lipid constituents of the Msm cell envelope (Etienne et al., 2005). The role of glycopeptidolipids (GPLs) in the aforementioned phenotypes is well established (Etienne et al., 2002; Recht & Kolter, 2001). To evaluate whether altered surface properties of the TopoI mutant could be due to an altered lipid profile, the GPLs from the WT, TopoI knock down and MstopoI complemented strain were isolated. Equal amount of dry cell mass was taken, and GPLs were isolated according to the established protocols (Khoo et al., 1996) and were subjected to thin layer chromatography (TLC). Analysis of the TLC data indicated the presence of characteristic spots of the polar GPLs in the WT strain (Patterson et al., 2000) which were significantly reduced in MsPptrtopoI (Fig. 3a). The influence of TopoI deprivation on the GPL biosynthesis was further evaluated by monitoring the expression of the mps and mmpl4b genes which are known to be involved in GPL biosynthesis (Billman-Jacobe et al., 1999). Transcriptional analysis suggested a significantly reduced expression of mps and mmpl4b in the TopoI depleted cells compared to the WT (Fig. 3b). The data indicate that the reduction in TopoI level affects the transcription of the genes involved in the lipid metabolic pathway resulting in altered surface properties of mycobacteria. TopoI-depleted strain acquires phenotypic drug resistance Altered surface properties and GPL levels of the mutant strain may influence the cell permeability which may affect the drug intake (Etienne et al., 2002) and thus alter the susceptibility of the cells to the drugs. Rifampicin and isoniazid, widely used anti-TB drugs were tested for their effect on the survival of the mutant. The analysis of the CFU for WT and MsPptrtopoI, indicated that the mutant strain exhibited reduced sensitivity to the drugs in 14

comparison to the WT (Fig. 4a). The increased survival of the topoI knock-down strain could be an attribute of low metabolic activity or altered permeability of the cell. The altered cell envelope could have a pronounced effect on the cell wall permeability barrier (Etienne et al., 2002). The cell permeability was determined by scoring EtBr uptake by the cells (Rodrigues et al., 2011). The mutant was compromised in EtBr uptake indicating the decreased cell permeability compared to the WT (Fig. 4b) which may contribute for the phenotypic drug resistance in the mutant. Reduction of TopoI leads to nucleoid de-compaction and altered supercoiling In a bacterial cell, the DNA topology is regulated by the combined action of topoisomerases and nucleoid associated proteins (NAPs) (Dillon & Dorman, 2010; Espeli & Marians, 2004). These two different kinds of topology modulators function in consort to organize the bacterial chromosome. In order to explore the effect of TopoI deprivation on nucleoid architecture, the nucleoid was examined by DAPI-fluorescence microscopy. The nucleoid of TopoI deprived cells was found in a de-condensed state across the bacterial cell, while the nucleoid of WT cells remained condensed (Fig. 5a). Since, the NAPs and topology modulatory proteins are responsible for the maintenance of nucleoid architecture, altered structure of the nucleoid might be a consequence of the perturbation in the expression levels of these proteins. To test this, the expression level of the major DNA topology regulators was monitored. In response to the reduction in TopoI level, the expression of DNA gyrase was also found to be reduced suggesting that co-ordinated expression of topoisomerases is required to maintain the topological homeostasis (Fig. 5b). The expression of TopoNM, an atypical type II topoisomerase found in Msm (22) was also reduced in the TopoI knock-down strain. Moreover, the transcript levels of the major NAPs (HU and Lsr2) were also reduced in the TopoI-depleted cells (Fig. 5c). It is 15

apparent that the imbalance in the TopoI levels brings about the change in nucleoid architecture as a consequence of the variation in the levels of other important topology modulators. Reduction in TopoI expression affects the supercoiling status of the genome (Pruss et al., 1982). To investigate the level of DNA supercoiling in the TopoI-depleted Msm cells, plasmid supercoiling was directly measured by isolating reporter plasmid (pMV261) and analyzed on chloroquine/agarose gel electrophoresis to resolve the various topoisomers. The increased accumulation of hyper-negative supercoiled plasmids in the TopoI-depleted cells compared to the plasmids isolated from the WT cells (Fig. 5d) indicated that the reduction in the TopoI level confers higher levels of negative supercoiling. Topol deprivation affects the proteome From the results presented, it is evident that the reduction in the TopoI level and hence perturbation of DNA topology results in various pleotropic effects affecting the physiology and genome organization of Msm. These variations in the properties of the mutant cells indicate global changes in the gene expression. Earlier studies in E. coli with TopoI mutant indicated that the increase in steady-state DNA supercoiling levels resulted in the alteration of abundance of various proteins (Steck et al., 1993). To investigate the consequences of TopoI deprivation on the global protein expression, 2-dimensional gel electrophoresis was carried out. The comparison of the proteome of WT and TopoI deprived cells demonstrated that multiple proteins were differentially expressed between the two strains (Fig. 6a and 6b). Few of the differentially expressed proteins were identified by peptide mass fingerprinting-mass spectrometry. These proteins are involved in diverse cellular pathways such as metabolic processes (FabG and Aldolase), transport (SSeC – a component of Secretion system III) and chaperonin (GroS) functions. The variation in the expression level of these genes was confirmed by their transcript 16

analysis (Fig. 6c). Altogether, it appears that the perturbation of steady-state supercoiling in Msm by reduction in TopoI levels could lead to the global changes in the gene expression. These results co-relate well with previous observations in E. coli where the mutation in the topoI affected the expression of various genes and operons (Steck et al., 1993; Sternglanz et al., 1981). TopoI deprivation affects RNAP distribution across the transcription units Aforementioned analysis of the TopoI mutant indicated the global changes in the cell physiology and gene expression. TopoI participates during the transcription process to remove the excess negative supercoils generated as a result of the advancement of RNAP along the template DNA (Broccoli et al., 2004; Masse & Drolet, 1999; Pruss & Drlica, 1986). Hence, the reduction of TopoI level would result in reduced relaxation activity, resulting in the accumulation of torsional stress across the genome and transcription units (TUs) which may affect the dynamics and the distribution of RNAP. To evaluate the influence of TopoI depletion on the RNAP distribution, we analyzed the occupancy of RNAP across the TUs of the genes affected in expression. The occupancy of RNAP was found to be reduced significantly on the Open Reading Frame (ORF) of the genes tested (Fig. 7a) suggesting the reduction in RNAP molecules in the elongation phase. To get further insights into the distribution of RNAP on the transcription units, the ratio (Traveling ratio) of RNAP occupancy on the gene promoter (Gene head) and ORF (Gene body) was determined (Reppas et al., 2006; Wade & Struhl, 2008). The increased Traveling ratio values indicated the accumulation of RNAP on the gene head and reduction in the elongating RNAP, implicating the aberrant elongation in the TopoI depleted state (Fig. 7b). Thus it appears that the reduction in TopoI levels affects the DNA topology of the genome leading to the inefficient transcription of several genes conferring various pleiotropic effects.

17

DISCUSSION A number of species of mycobacteria including Mtb possess a single type I topoisomerase and a DNA gyrase to take care of the entire burden of topological inter-conversions in the cell. In contrast, a large number of actinomycetes, including Msm have additional topoisomerases. In addition to an extra orphan GyrB (Jain & Nagaraja, 2002) and an unusual Type II relaxase termed as TopoNM (Jain & Nagaraja, 2005), the Msm genome encodes a Type IB (MSMEG_1784) topoisomerase (http://mycobrowser.epfl.ch/smegmalist.html) similar to the one found in poxviruses. Such a type IB enzyme is shown to possesses DNA relaxation activity in Deinococcus radiodurans (Krogh & Shuman, 2002). The presence of additional topoisomerases in the genome suggests their role in assisting the principal enzyme in DNA relaxation and maintaining the topological homeostasis. This in turn may imply the functional redundancy of DNA relaxation enzymes in Msm, other fast growing mycobacteria and members of the genus Streptomyces. Thus it was important to investigate whether TopoI is absolutely essential for Msm survival or the cells could cope up with the “back up” relaxation machinery when the TopoI levels were depleted. The results presented in this study demonstrate that TopoI in Msm is essential for cell growth and proliferation. The variation in the TopoI levels conferred altered colony morphology and phenotypic characteristics. Additionally, the depletion in the enzyme levels resulted in altered expression of other topology modulatory proteins and the consequent diffused nucleoid revealed the importance of TopoI in the maintenance of nucleoid architecture. In contrast to the TopoI mutant of Msm which acquires phenotypic drug resistance, Mtb TopoI mutant did not show drug resistance (23). Instead, the Mtb strain exhibited enhanced susceptibility to novobiocin and INH upon depletion of TopoI with ATc (Ahmed et al., 2014). These seemingly opposing results are actually due to the different levels of TopoI in the two 18

species. Analysis of the TopoI level in the mutant strains of both the organisms (in the absence of ATc) indicated the reduced levels of TopoI in Msm mutant compared to the WT strain whereas in Mtb mutant the levels were higher than WT. These results suggest variation in the expression levels of the enzyme in the two species due to the differences in their respective promoter activity. Thus the alteration of TopoI level or supercoiling alterations in different organisms may have different effects as illustrated previously in Salmonella and E. coli (Cameron et al., 2011). Notably, two clinical strains of Salmonella having different mutations in gyrase genes exhibited diverse supercoiling and phenotypic properties suggesting that varied level of topoisomerase activities can influence the physiology of the organism differentially (51). TopoI depleted Msm exhibited growth defect and pleotropic phenotypes (Schematic in Fig. 8). The mutant acquired altered physiology, metabolism and drug resistance due to alteration in TopoI levels or consequent altered genome supercoiling influencing diverse pathways leading to global changes. The importance of topoisomerases in the maintenance of genome supercoiling and thus fine tuning of the expression of various genes involved in adaptation, stress and virulence has been studied in various systems. In Salmonella, expression of two Type III secretion systems (TTSS) involved in adaptation and pathogenesis inside the host showed sensitivity to genome supercoiling (Galan & Curtiss, 1990; T et al., 2006). In E. coli switching of fimbriae gene was TopoI dependent (Dove & Dorman, 1994). Similarly, the mutation in topoI led to reduced expression of recA in E. coli conferring increased UV sensitivity (Urios et al., 1990). Moreover, deletion of topoI reduced the expression of the fooB regulator required for the expression of fimbriae F165 (1) needed for the virulence and pathogenesis of E. coli 4787 (O115:KV165) (Tessier et al., 2007). Also, a mutation in topoI suppressed the leu-500 promoter mutation (a supercoiling sensitive promoter) leading to the enhanced expression by perturbing 19

the topology of regulatory DNA elements (Pruss & Drlica, 1985). These studies suggest that the alteration in topoisomerase activities may lead to alteration in global gene expression and various phenotypic consequences. A detailed characterization of a DNA gyrase mutant in Salmonella suggested a change in global genome supercoiling and resistance to various drugs (Webber et al., 2013).The transcriptome analysis of the mutant revealed the altered expression of the genes involved in export, stress and metabolic pathways (Webber et al., 2013) conferring numerous phenotypic changes. In S. pneumonia inhibition of DNA gyrase resulted in the activation of stress responsive, virulent genes and simultaneous down regulation of several housekeeping genes involved in cell metabolism and growth (Ferrandiz et al., 2010). Thus previous and present studies show that the perturbation in the topoisomerase activity confers global changes in the bacteria leading to altered phenotype and drug sensitivity. The diffused nucleoid architecture of the Msm mutant indicated a perturbation of the genome supercoiling. The supercoiling level of the genome may influence the initial binding of RNAP holoenzyme and subsequent activation of specific sets of promoters leading to the global changes in the gene expression. An earlier study in E. coli suggested that the binding of stationary phase specific σ 38 to the target gene was favored on a template with low super-helical density while the binding of house-keeping σ 70 was optimal with the negative supercoiled DNA (Kusano et al., 1996). The altered supercoiling of the bacterial genome may potentially re-wire the distribution of different species of holoenzymes on the transcription units leading to the change in global gene expression profile. Moreover, the reduction of TopoI activity in the bacteria would lead to the accumulation of excessive negative supercoiling that may impede the movement of RNAP and its subsequent distribution on the transcription units (TUs). The occupancy profile of RNAP on the genes affected in expression in the Msm mutant revealed the 20

varied distribution of the RNAP on the TUs. The accumulation of RNAP on the promoter region of the TUs could be a result of excessive negative supercoiling (torsional strain) which in turn may retard the entry of RNAP into the elongation phase. These alterations could impact the global transcription profile of the cell leading to the changes illustrated. Similar observation was reported in a yeast Topo II mutant where the distribution of RNAP on TUs was affected upon depletion of Topo II activity (Joshi et al., 2012). A mutation in the gene or reduction in topoisomerase activity in neuronal cells resulted in the accumulation of RNAP on the promoter region of TUs (King et al., 2013). Although different from Mtb in various respects, Msm is often used as a surrogate host for a variety of studies (Chaturvedi et al., 2007). The use of Msm as a surrogate for Mtb for drug screening and genetic studies is a matter of debate (Ramon-Garcia et al., 2013). However, in the present study, the ability of MttopoI to functionally complement Msm topoI knock down strain offers an opportunity to carry out genetic studies to better understand the function of MttopoI and for screening the specific inhibitors against the enzyme. Given the faster growth rate of Msm, the strain would expedite the studies related with the in vivo function of Mtb TopoI and to exploit it as a drug target. The importance of TopoI, the first described topoisomerase originally named as omega protein can be appreciated by the vast body of information on its mutational analysis in E. coli. Early genetic studies revealed the requirement of TopoI for the E. coli growth. The deletion of TopoI was found to be lethal in the absence of any compensatory mutations in DNA gyrase (DiNardo et al., 1982). The direct evidence of TopoI essentiality came from the study using a temperature sensitive TopoI mutant where the inactivation of TopoI at 42°C led to the arrest of cell growth (Zumstein & Wang, 1986). Thus, in spite of having additional DNA relaxation 21

enzymes (Topo III and Topo IV), the TopoI function is of critical importance in E. coli. In contrast, the TopoI null mutant of S. Typhimurium and Shigella flexineri were found to be viable even in the absence of any compensatory mutations (Ni Bhriain & Dorman, 1993; Trucksis et al., 1981)

- proteobacteria, thus seem to have varied impact on growth

and survival when their respective TopoI is depleted. In contrast, in both the mycobacterial species TopoI appears to be indispensable for cell survival. To conclude, TopoI is essential in different species of mycobacteria irrespective of whether they possess a sole enzyme or have additional DNA relaxation enzymes. TopoI-depleted Msm strains may offer an unique opportunity and advantage to study the Mtb counterpart. The importance of TopoI seems to be varied across the eubacterial species ranging from indispensability to having specific dedicated role. Nevertheless, the present and earlier studies emphasize its critical importance in maintenance of topological homeostasis. Perturbation in TopoI expression conferred various phenotypic effects as a consequence of altered distribution of transcription machinery on TUs. Analysis of the global transcriptome and RNAP distribution on the TUs of the TopoI mutant of mycobacteria may facilitate the further understanding of the connection between genome supercoiling, gene expression and phenotypic characteristics in this group of organisms. Acknowledgements The authors thank the members of VN laboratory for valuable suggestions and critical reading of the manuscript. W.A. is a recipient of Shyama Prasad Mukherjee fellowship of Council of Scientific and Industrial Research (CSIR) and V.N. is a J.C. Bose Fellow of the Department of Science and Technology, Government of India. The work is supported by grants from 22

Department of Biotechnology, Government of India and the European Community's Seventh Framework Program (MM4TB).

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FIGURE LEGENDS Figure 1. Optimal TopoI level is required for normal growth of M. smegmatis. (a) Expression analysis of TopoI by immunoblotting using the TopoI specific antibody demonstrates the effect of anhydrotetracycline (ATc) on the TopoI expression; (b) TopoI conditional mutant requires additional supplements for growth. The cultures were inoculated in 7H9 broth with or without ADC (as indicated) and incubated at 30°C; (c) The growth of WT and MsPptrtopoI in liquid culture in the presence of various concentrations of ATc; (d) Complementation of TopoI mutant by ectopic expression of MstopoI or MttopoI. Exponential phase cultures were inoculated into the fresh 7H9-ADC media (with or without ATc) to 0.05 O.D.595nm .The cultures were incubated at 30°C and growth was monitored by measuring the O.D.595nm at constant time intervals. The graph obtained is the average of three independent experiments. Figure 2. Altered phenotype of MsPptrtopoI. (a) Colony morphology was demonstrated by growing the colonies on 7H9 agar for 8 days; (b) Sliding motility was determined on 0.4% agar; (c) Biofilm formation: The quantitation of biofilm formation ability was carried out using crystal violet staining; (d) Pellicle formation was seen in the static culture without Tween 80. Figure 3. Expression of glycopeptidolipids (GPLs). (a) Exponential phase cells were harvested and equal mass of WT and mutant cells was taken and processed for GPL isolation. The isolated lipids were separated on silica TLC sheets and developed by 10% H2SO4 and 1% Naphthol; (b) Analysis of the expression of the genes involved in GPL biosynthesis by RT-PCR. The relative expression of the genes in mutant with respect to the WT is represented in the bar graph. Error bars represent the standard deviation obtained in two independent experiments.

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Figure 4. Cell wall permeability and altered drug susceptibility of the mutant (a) The survival of the MsPptrtopoI and WT after 9 h treatment with Rifampicin (RIF) and Isoniazid (INH) was estimated by determining the colony forming units (CFU). ND-No drug; (b) The cellular permeability of the mutant strain was compared with the WT strain by determining the uptake of EtBr using fluorometry. Figure 5. Perturbation of DNA topology modulators and nucleoid decompaction. (a) Exponential phase cultures of WT and conditional mutant were treated with ATc (100 ng/ml), stained with DAPI and visualized by fluorescence microscopy; (b) Immunoblot analysis of TopoI and GyrB expression in conditional mutant; (c) Transcript expression analysis of major topology modulators under TopoI deprived condition in the mutant; (d) pMV261 plasmids isolated

from

the

exponential

phase

mycobacterial

cells

were

subjected

to

the

chloroquine/agarose gel electrophoresis. Figure 6. Identification of differentially expressed proteins in TopoI-depleted M. smegmatis. Silver stained 2DE gels of (a) WT and (b) MsPptrtopoI. Isoelectric focusing of cell lysates was carried out on 17 cm IPG strip of pH 3-10 and a 14% SDS PAGE was used to resolve in the second dimension. The arrows indicate the differentially expressed protein spots in the two strains which were subjected to mass spectrometry analysis; (c) RT-PCR validation of the genes identified by 2D-MS. Change in gene expression is represented as a ratio of transcripts detected in MsPptrtopoI and the transcripts detected in WT. Figure 7. Distribution of RNAP on transcription units (TUs). The occupancy of RNAP was determined by Chromatin immunoprecipitation (ChIP) using RpoB specific antibody followed by detection of specific region of TUs by RT-PCR; (a) ChIP-RT PCR analysis of the RNAP 30

occupancy on the ORF of the genes affected in transcription (b) Determination of traveling ratio of RNAP on the affected genes. The traveling ratio was determined by calculating the ratio of the occupancy of RNAP on the gene promoter region and the occupancy on the ORF. Figure 8. Perturbation in TopoI level leads to the changes in various phenotypic characteristics in M. smegmatis.

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