Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species (ROS) but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how Mtb manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in Mtb. Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Mtb. Disruption of rv0158 (rv0158 KO) impaired growth, redox balance, respiration, and metabolism of Mtb on glucose but not on fatty acids. Importantly, rv0158 KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of rv0158 enhanced the sensitivity of Mtb to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, rv0158 KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in Mtb. Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), a disease that results in approximately 1.6 million deaths annually (Mtb requires oxygen (O2) for growth  as metabolic by-products when O2 adventitiously interacts with and extracts an electron from quinones, flavins, and transition metal centres distributed throughout the cell , nitric oxide (NO), their reaction product peroxynitrite (ONOO.), and hypochlorite stress , superoxide dismutase (Fe-SodA), thioredoxin reductase (TrxB2), alkyl hydroperoxide reductase (AhpC), and redox buffers- thioredoxin (trxC), and ergothioneine  in millimolar amounts  concentrations of exogenous ROS and RNI , we found that heterogeneity in MSHE of Mtb drives multidrug tolerance inside macrophages  and redox regulators are required to maintain MSHE, ergothioneine biogenesis, NAD+/NADH poise, and to counter the lethal action of antibiotics in Mtb  and exploited FACS to reliably separate mutants displaying a shift from basal to oxidative MSHE during aerobic growth. We then developed a next-generation sequencing (NGS) and analysis pipeline for constructing a system-level network of genetic factors essential for maintaining redox homeostasis under aerobic growth in Mtb. We established the effectiveness of our strategy by discovering the role of an unknown transcriptional regulator encoded by rv0158 in integrating the metabolism of carbon source with redox balance and bioenergetics in Mtb.In this work, we repurposed transposon mutagenesis- deep sequencing (TnSeq) by combining it with the Mrx1-roGFP2 redox biosensor to discover novel pathways controlling basal 280±5 mV . We buil280±5 mV  in Mtb e5 independent insertion events was generated in Mtb-roGFP2 (600 nm = 0.6–0.8), and genomic DNA was isolated for deep sequencing of transposon insertion (TnSeq) by Illumina technology. Using Transposon-directed insertion-site sequencing  comprising >10b-roGFP2 . The poo TraDIS; , we iden TraDIS; . Next, wDIS; Mtb . Eighty-b genome . TherefoMSHE as the MSHE of mid-log phase Mtb grown in 7H9+ADS under standard aerobic culture conditions   of the library using flow-cytometry . To set the gates for sorting MSH-Eoxidized Tn-mutants by flow-cytometry, we treated wild-type (WT) Mtb H37Rv expressing Mrx1-roGFP2 with an oxidant, cumene hydroperoxide (CHP). Treatment with 10 mM CHP for 5 min maximally increased the 405/488 ratio  . Mtb exh2±4.7 mV . We aimeytometry . To sort88 ratio , indicatstrategy . TherefoMSHE under aerobic culture conditions without exposure to exogenous oxidants , hydrogen peroxide (H2O2), and menadione  after 8 hr of treatment , redox-sensor (whiB6), cysteine metabolism , antioxidant enzymes , Fe homeostasis , sufR, redox-regulated chaperone , were overrepresented in the MSH-Eoxidized Tn-mutant pool , oxidized DNA and lipid repair , membrane integrity, RNA processing, and amino acid metabolism were also part of Mtb redoxosome . Both Mtb CDC1551 and Mtb H37Rv displayed identical basal MSHE   or a target gene, and a directed edge indicates the transcriptional activity of a target gene by a TF. Further, the largest component of 102 genes and 139 edges in the redox-homeostasis network can be organized into three hierarchical levels with 6 TFs in the top, 6 TFs in the middle, and 90 target genes in the bottom  that helps  E. coli  and S. crevisiae . We, therk (TRN) . A similrk (TRN) , adaptatrk (TRN)  and to hrk (TRN)  in Mtb. e bottom .dosR responsive to O2/NO/CO/H2S  by qRT-PCR , RNA was harvested and subjected to RNA-seq. We identified 232 genes that were differentially expressed  in the absence of rv0158 , catalase-peroxidase (katG), chaperones , epoxide hydrolase (ephA), and DNA-repair genes  were upregulated  and nitrosative stress . In contrast, transcriptional changes associated with acidic pH, known to induce reductive MSHE in Mtb  to identify f rv0158 . We valif rv0158 . Consistegulated . SupportH in Mtb  and hypocriptome . CollectfadA, fadB, echA7, fadE12, fadE13, fadE20, fadD7, accA2, accD2, fadE23, fadE24, fadD9, fadD22), propionate metabolism   were downregulated in the KO strain  and a simultaneous accumulation of fatty acid methyl esters  in rv0158 KO as compared to WT Mtb and rv0158 Comp   were sigO strain . Agreein158 Comp . A decreeability . Consistsoniazid  and ethi158 Comp . Altogetrv0158 KO. To examine the role of rv0158 in utilizing fatty acids as nutrients by Mtb, we measured the growth of WT Mtb, rv0158 KO, and rv0158 Comp in 7H9 medium supplemented with either 0.3% glucose or host-relevant fatty acids  as a sole carbon (C) source , which alleviate propionate toxicity  source . Both ab) source . The defalmitate . The mettoxicity , were si158 Comp .MSHE. The rv0158 KO was significantly more oxidized (MSH =E –266.3 ± 5) as compared to WT Mtb (MSHE = –279.4 ± 3.5) and rv0158 Comp (MSHE = –277.4 ± 2) on glucose (Mtb cells growing on acetate or propionate or cholesterol uniformly exhibit a reductive shift in MSHE (−285 to –305 mv) compared to glucose (–279 mV), agreeing with fatty acids being a more reduced C substrate than glucose (MSHE of rv0158 KO showed a reductive shift and recovered to WT Mtb levels (−285 to –305 mv) on acetate/propionate/cholesterol  and OCR (oxygen-consumption rate) between WT Mtb and rv0158 KO were measured by sequentially injecting a C source and membrane potential uncoupler (CCCP) in the XF microchamber. CCCP depolarizes the cell membrane and stimulates maximum respiration in Mtb. Fold changes were measured by comparing the OCR or ECAR readings after glucose or CCCP addition. When glucose at 0.3% was injected, the %OCR and %ECAR were ≈ two fold less in rv0158 KO than in WT Mtb and rv0158 Comp (rv0158 KO (rv0158 KO fully recovered to WT Mtb levels when 0.3% acetate or 0.3% propionate was provided as the sole C source (rv0158 KO more than WT Mtb and rv0158 Comp in the propionate-containing medium (6 cells/well) were used for these assays, the changes in OCR and ECAR were likely due to altered bioenergetics of the rv0158 KO strain.Variable growth on fatty acids versus carbohydrates directly influences the bioenergetics of Mtb . We test158 Comp . As expev0158 KO . ImportaC source . Furtherg medium . Since arv0158 KO to propionate as a C source, we performed liquid chromatography-mass spectrometry (LC-MS) metabolomics following exposure of WT Mtb and rv0158 KO to 0.3% glucose or 0.3% propionate. The consumption of propionate requires overlapping reactions encompassing the TCA cycle, methyl citrate cycle (MCC), and glyoxylate cycle  or marginally accumulated (e.g. fumarate and glyoxylate) in propionate-grown rv0158 KO  in WT Mtb, rv0158 KO, and rv0158 Comp growing on glucose or propionate by qRT-PCR. Consistent with the RNA-seq data, we observed increased expression of MCC genes in rv0158 KO compared to WT Mtb on glucose (rv0158 KO compared to WT Mtb (Mtb mitigates propionate toxicity via the biosynthesis of PDIM (phthiocerol dimycocerosates)  . We measo WT Mtb . Howeveropionate . The exp158 Comp . Overallthe rv0158 gene has been classified into the TetR family of transcriptional regulators (TFTRs) (H-4α(3-propanoate)–7aβ-methylhexahydro-1,5-indanedione- coA (HIP-CoA)) responsive, TetR-family transcription factor KstR2   to identify ligands for Rv0158. We tested various metabolites generated during the catabolism of long-chain fatty acids, propionate, and cholesterol  . In sili2*10–13) . Histidi2*10–13) . Since rlesterol . We founlesterol . Howeverlesterol .Mtb. To examine this, we performed ChIP-Sequencing (ChIP-Seq) using an anhydrotetracycline (ATc)-inducible flag-tagged Rv0158 . Further classification of these genes indicates that Rv0158 binds to several genes involved in methyl-branched lipid biosynthesis , lipid transporter , biotin synthesis (bioF2), and surface-associated proteins .Since methylmalonyl-CoA is a crucial intermediary metabolite regulating propionate flux into MCC, methylmalonyl pathway, and methyl branched lipids , we propq value <0.0001). Out of 32 genes, 24 genes overlap with glucose condition. The promoter regions of the remaining eight genes Rv0158 exclusively bound in propionate- grown Mtb were part of the glyoxylate cycle and MCC  and fadB2 , B represents G/T/C, and M represents A/C, enrichment e value = 2.6*10E. coli  as C source (Mtb) in the knockout strain when growing with fatty acids as C source  and second-line anti-TB drugs (moxifloxacin and bedaquiline) by monitoring the resazurin reduction to resorufin using resazurin microtiter assay (REMA). In the absence of antibiotics, we did not see any difference in the extent of resazurin reduction to resorufin among the untreated Mtb strains  than WT Mtb and rv0158 Comp   and identified genetic factors required for maintaining basal redox balance in Mtb. We noted that during enrichment cycles, few unfit Tn-mutants exhibiting overwhelming oxidative MSHE  might have been outcompeted by relatively fit Tn-mutants in the Mtb redoxosome. Additionally, certain antioxidant enzymes  are essential (Mtb.While regimen) . These fssential  and, thessential . Interesssential . Future rv0158, as a critical node with the highest betweenness centrality in the hierarchical redox-homeostasis network. Interestingly, rv0158 is one of the key transcriptional regulators that genetically interact with ctpC (sodA hypomorph) in a network to impart oxidative stress resistance during infection in mice  increased catabolism which then leads to the accumulation of chemically reactive and biologically intoxicating metabolites, such as propionate, glyoxylate, or fumarate  and anabolic  pathways of Mtb.ty acids . β-oxidaids rv0158 KO grew slow in response to glucose, the growth of the mutant was either comparable on fatty acids  or better (propionate) than WT Mtb. Since propionate stunts the growth of WT Mtb but not of rv0158 KO, we begin understanding the role of rv0158 in propionate metabolism. It is known that propionate  is lethal to Mtb if allowed to accumulate  suggest that the mutant is better equipped to prevent accumulation of toxic intermediates when metabolizing propionate. Another consequence of growth on propionate is the undesirable accumulation of NAD(P)H pool, resulting in intrabacterial reductive stress (MSHE of Mtb on propionate suggests that Mtb utilizes mycothiol as a sink to dissipate NAD(P)H-reductive stress. Reductive stress is also mitigated by NADPH-dependent incorporation of propionyl moieties into cell wall polyketide lipids  and triacylglycerol (TAG) (rv0158 KO displayed higher expression of PDIM biosynthesis genes-cluster (MSHE (While cumulate . Similarcumulate . The rele stress . A reducol (TAG) . Propion-cluster , a more er , impaired resistance to redox stress, antibiotics, and persistence defect in mice. The in vivo phenotype of rv0158 KO is consistent with the fact that Mtb persistence relies upon efficient metabolization of glucose, co-catabolism of multiple nutrients, and resistance to oxidative/nitrosative stress in mice  required for the biotin-dependent conversion of propionyl-CoA to methylmalonyl-CoA are highly induced in rv0158 KO . These findings suggest a possible regulatory loop between biotin synthesis, AccA2/D2-dependent methylmalonyl-CoA formation, and propionate metabolism, where Rv0158 may act as an important link between these pathways. The vitamin B12 (Vit-B12) dependent methylmalonyl-CoA pathway is another means by which propionate toxicity can be alleviated in Mtb  in d in Mtb . While wprp operon is carefully controlled by PrpR. Under propionate-rich conditions, PrpR binds to propionyl-CoA and induces the expression of itself, prp operon, and icl1 to catabolize propionate by MCC  and metabolic  pathways in Mtb.The transcription of the e by MCC . Howeverand icl1 . The exponyl-CoA . TherefoMtb, and how this association affects direct interaction with DNA and gene expression. Also, the lack of Rv0158 may induce a physiological state in Mtb that is more tolerant to toxicity caused by fatty acids such as propionate. For example, cAMP homeostasis is associated with modulating redox balance, respiration, fatty acid uptake, and fatty acid toxicity in mycobacteria  to direct the flux of excess fatty acids away from catabolism to lipid anabolism  in Mtb  in coordinating fatty acid metabolism.Although our data suggest that Rv0158 likely senses methylmalonyl-CoA and regulates the expression of pathways associated with propionate metabolism, several issues remain to be addressed. It remains unknown if methylmalonyl-CoA is perceived as a metabolic signal by Rv0158 in bacteria . Moreoveopionate . Other eopionate  as we obnabolism . These aenvelope . We obse) in Mtb . TherefoMycobacterium smegmatis mc2155 (Msm) and Mycobacterium tuberculosis H37Rv (Mtb) were kind gifts from Dr. Kanury V.S. Rao . Strain Mtb-Mrx1 roGFP2 was generated by transforming Mtb H37Rv with an E. coli-mycobacterial shuttle vector, pMV762 . Mtb H37Rv mshA KO was a kind gift from Prof. William R. Jacobs Jr . Wild-type Mtb CDC 1551 and the Mtb CDC1551 transposon mutants were collected from BEI resources (https://www.beiresources.org/). Plasmid pexCF-rv0158 was a kind gift from Prof. David Sherman ., pMV762 . PlasmidAll strains were grown in 7H9 broth  supplemented with 0.2% glycerol, 0.1% Tween-80, and ADS , 0.2% dextrose, and 0.085% NaCl) with shaking at 180 RPM in a rotary shaker incubator  or on 7H11 agar  supplemented with ADS or OADC . and 0.004% catalase  at 37 °C. Bacterial strains expressing Mrx1-roGFP2 were grown in media containing hygromycin. Hygromycin  and kanamycin  were used at a final concentration of 50 µg/mL and 25 µg/mL, respectively. To measure bacteria’s response to oxidative stress, assays were performed in the 7H9 medium supplemented with a catalase-negative supplement ).Mtb strains were precultured to OD 600 nm of ≈1.0, washed with 1 X PBS, and resuspended in fresh medium media containing 0.47% 7H9, 0.5% albumin, 0.085% NaCl, and 0.05% tyloxapol  supplemented with 0.3% (w/v) D-glucose  or 0.3% (w/v) sodium acetate , 0.3% (w/v) or sodium propionate  or 0.02% (w/v) cholesterol  or 200 μM sodium palmitate , followed by incubation at 180 RPM in a rotary shaker incubator.Protocol for carbon-utilization experiments was followed as described in previous studies . BrieflyMtb H37Rv-Mrx1 roGFP2 strain was grown till OD600 nm ~1. The cells were washed twice with wash medium (7H9 broth +5% glycerol) and resuspended in mycobacteriophage buffer . These phage-competent cells were infected with phAE180 (1010–1011 PFU/mL) at MOI of 1:10 for 3 hours at 37 °C followed by the selection of phage-transduced Mtb cells on 7H11+OADC plates containing hygromycin and kanamycin and incubated at 37 °C for 4–5 weeks.>105 colonies were scrapped, pooled together, and stored at –80 °C as frozen glycerol stocks.ATGGCAGAACAAACTGAG3’ and TM4-ScaR: 5’GAATTGGTGTTGCCGTT G3’. TraDIS (TnSeq) protocol was utilized to identify the site of mutation (insertion by transposon) in the library of Mtb-transposon mutants  were obtained for two biological replicates, each for the Tn-Library and MSH-Eoxidized Tn mutant pool of Mtb as.fastq files. The reference genome sequence (.fna) and annotation (.gff) files for the same strain were downloaded from the NCBI ftp website (https://ftp.ncbi.nlm.nih.gov/). The reads were mapped to the Mtb H37Rv genome (accession number: NC_018143.2) using Burrows-Wheeler Aligner  in either of the pipelines were considered enriched for insertions in MSHE- oxidized Tn mutant pool relative to the input Tn Lib. The union of over-represented genes derived from DESeq2 and edgeR is considered ‘Mtb redoxosome’. The TraDIS data has been submitted to NCBI’s BioProject .Genomic DNA for TraDIS (TnSeq) protocol was isolated as previously described . The qua mutants . Briefly mutants . PCR-enrner BWA; . The rawner BWA;  and DESener BWA;  pipeline600 nm = 0.6–0.8 in OADC-supplemented 7H9 media at 37 °C, 180 rpm. Prior to experiments, cells were harvested and resuspended in 7H9 media containing ADS. Flow-cytometry using a BD FACS Aria flow cytometer equipped with the 405 nm and 488 nm lasers was performed to determine the ratio of Mrx1-roGFP2 fluorescence within Mtb cells at 510 nm emission. Ratiometric changes (Ex: 405/488 nm) upon 10 mM cumene hydroperoxide (maximum biosensor oxidation) and 20 mM dithiothreitol (maximum biosensor reduction) treatment to WT Mtb were utilized to gate ambiently MSHE-oxidized or MSHE-reduced Mtb transposon mutants. 10,000 events per sample were analyzed. For enriching this population, cells were sorted using BD FACS Aria with a four-way purity sort setting. The oxidized MSHE of sorted cells was confirmed by post-sort analysis and plated on 7H11 supplemented with OADC and allowed to grow at 37 °C for 4–5 weeks. The intracellular MSHE measurements were done as described previously (Bacteria were grown to ODeviously .600 nm = 0.6). Genomic DNA was isolated from each sample. 8-OH deoxyguanosine species were quantified using ELISA according to the manufacturer’s instructions .Bacterial cultures were grown to the mid-log phase (OD600 nm = 0.6). Lipid hydroperoxides were quantified from cell pellets using FOX2 reagent . Bacterial clumps were removed by centrifugation at 200 g for 5 minutes. The single-cell suspension was then set at 0.1 OD600 nm. Oxidants , cumene hydroperoxide , diamide , menadione  and Diethylenetriamine/nitric oxide adduct  at indicated concentration were added to the culture, and CFU was determined after 8 or 24 hr of treatment. In vitro growth kinetics of bacterial strains were determined by removing 200 μL aliquots at various intervals, serially diluted, and plated on 7H11 agar for CFU enumeration. The killing of bacterial strains in the presence of anti-TB drugs , Rifampicin , moxifloxacin  and bedaquiline ) was quantified by treating bacteria with antibiotics as described in Resazurin Microtiter Assay (REMA) protocol . Approximately 1×105 bacteria per well were added in a total volume of 200 µL of 7H9+ADS medium. Wells lacking Mtb served as a control. Additional controls consisted of wells containing cells (growth control). After 5 days of incubation at 37 °C in the presence of anti-TB drugs, 30 µL of 0.02% resazurin  was added, and plates were incubated for an additional 24 hr. Fluorescence intensity was measured using a SpectraMax M3 plate reader (Molecular Devices) in the bottom-reading mode with excitation at 530 nm and emission at 590 nm. Percent inhibition was calculated from the relative fluorescence units compared with an untreated control culture; MIC was taken as the lowest drug concentration that resulted in at least a 90% reduction in fluorescence compared to untreated growth control.MIC was determined by a resazurin microtiter assay (REMA) using 96-well flat-bottom plates . Mtb strBacteria were grown in OADC-supplemented 7H9 media at 37 °C, 180 rpm. Prior to experiments, cells were harvested and resuspended in 7H9 supplemented with ADS. These cells were treated with 12.5 µM cumene hydroperoxide, and the Mrx1-roGFP2 biosensor ratio was measured by flow-cytometry to investigate the redox response of cells as a function of time. 10,000 events per sample were analyzed.600 nm ≈ 0.6). 25 mL GTC buffer , 0.5% sarcosyl, 0.5% Tween-80, 1% β mercaptoethanol (freshly added)) was added to 10 mL of bacterial culture, mixed, and cells were immediately harvested. Total RNA was extracted as described previously using MP Biomedicals FastRNA Pro Blue Kit  and QIAGEN Rneasy Kit .Bacterial strains were grown to the mid-log phase (ODMtb Transcription Regulatory Network (TRN) consisting of 156 Transcription factors (TFs), 3679 target genes, and 15,980 protein-DNA interactions based on Chromatin Immunoprecipitation followed by Next-generation Sequencing (ChIP-Seq) data (Mtb TRN wherein both the TF and the target gene of an interaction are part of the redoxosome of 368 genes which are overrepresented in the MSHE- oxidized Tn mutant pool. The redox-homeostasis network has three connected components with a dominant largest component comprising of 102 genes (including 12 TFs) and 139 edges (We used the eq) data  to genereq) data . This re39 edges . Moreove39 edges  for the 39 edges .rv0158-ORF were amplified and cloned upstream and downstream, respectively, of the loxP-hyg-gfp-loxP  cassette in the mycobacterial suicidal vector pML523 . The complete construct of ‘flanking sequences along with the hyg-gfp DNA sequence’ was cloned into the pRSF-duet vector (Clontech). UV-treated pRSF-rv0158 was electroporated into WT Mtb H37Rv for allelic exchange as described previously  by antibiotic selection and quantitative RT-PCR (qRT-PCR). The knockout strains were unmarked using the pCre-Zeo-SacB plasmid . For generating complemented strains, rv0158 ORF and upstream 400 bp promoter sequence were cloned into pCV125 . Cells were harvested by centrifugation (4200 g for 5 min) and resuspended in 100 µL of growth medium. As per the manufacturer’s instructions, CellROX Deep Red  was added to a final concentration of 5 µM, and cells were agitated on a rocker (Biobee Tech) for 30 min at 37 °C. After incubation, cells were washed to remove residual dye by centrifugation (4200 g for 5 min). Cells were resuspended in 300 µL phosphate-buffered saline (pH 7.4) and then fixed by adding 4% paraformaldehyde (PFA) for 1 h at room temperature. Fluorescence of 10,000 events per sample was measured at a fixed emission (670 nm) after excitation with a red laser (640 nm) using a BD FACSVerse Flow cytometer (BD Biosciences). 10,000 events per sample were analyzed.Bacterial strains were grown to the mid-log phase , which is based on TUNEL (TdT-mediated dUTP-X nick end labeling) assay (600) were taken, washed once by centrifugation, and fixed in 2% paraformaldehyde (PFA). PFA was removed by washing cells, followed by resuspension in 2% sodium dodecyl sulfate (SDS) and a second wash by centrifugation. DNA double-strand breaks were labeled in 100 μl of TUNEL reaction mix for 3–4 hr. Cells incubated with label solution only  were used as negative controls. Fluorescence was measured at a fixed emission (585 nm) after excitation with green-yellow laser (561 nm) using a BD FACSAria Flow cytometer . A total of 10,000 events were acquired per sample.DNA damage was measured by using g) assay . An equa600 nm ≈ 0.6). Total RNA was extracted as described above. Ribosomal RNA (16 s and 23 s rRNA) was depleted from RNA samples using MICROBExpress Bacterial mRNA Enrichment Kit . RNA was quantified using Qubit 2.0 Fluorometer (Invitrogen). rRNA depleted RNA (≈ 15 ng per sample) was fragmented and then used to prepare the library using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina  and NEBNext Multiplex oligos for Illumina , according to the manufacturer’s protocol. The fragment-size distribution, uniformity, and quality were examined using a high-sensitivity DNA Chip in Agilent Bioanalyzer (2100 High sensitivity). Two nM of cDNA libraries were pooled together and sequenced using the HiSeq2500 platform (Illumina).Bacterial strains were grown to the mid-log phase (ODMtb H37Rv were downloaded from the NCBI ftp website. The reads were mapped to the Mtb H37Rv genome (accession number: NC_000962.3) using Burrows-Wheeler Aligner (BWA) . Genes with absolute fold change ≥1.5 or≤–1.5 and FDR <0.05 were considered differentially expressed in comparative groups. Raw data has been submitted to NCBI’s Gene Expression Omnibus .Raw reads  were obtained for two biological replicates as.fastq files. The reference genome sequence (.fna) and annotation (.gff) files for the same strain- er (BWA) . The rawMtb cultures were briefly (one day) incubated in 7H9 medium containing the non-metabolizable detergent tyloxapol and lacking ADS or a C-source. These cultures were then passed 10 times through a 26-gauge syringe needle followed by centrifugation at 100 g for 1–2 min to remove clumps of bacterial cells. The resulting single-cell suspensions of bacteria at 2×106 cells/well were placed in the bottom of wells of a Cell-Tak -coated XF culture plate (Agilent/Seahorse Biosciences). To confirm that an equal number of cells were seeded on the plate, Mtb strains were plated on 7H11 agar, and CFU was enumerated after 4 weeks. Measurements were performed using a Seahorse XFp analyzer (Agilent/Seahorse Biosciences) with cells in an unbuffered 7H9 growth medium (pH 7.45 lacking monopotassium phosphate and disodium phosphate). OCR and ECAR measurements were recorded for ∼21 min  before the addition of either glucose  or sodium acetate (0.3% w/v) or sodium propionate (0.3% w/v) as a C-source, which was delivered automatically through the drug ports of the sensor cartridge . Carbonyl cyanide 3-chlorophenylhydrazone  at 10 μM final concentration was similarly added through drug ports as indicated in figures. Changes in OCR (% OCR) and ECAR (% ECAR) readings triggered by C-source or CCCP were calculated as a percentage of the third baseline reading for OCR and ECAR taken before C-source injection.To measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), log-phase Mtb was performed as previously described  and incubating for another 24 hr. Bacteria were harvested after washing with 1 X PBS and sterilized by autoclaving. To the cell pellets, 2 mL of tetra-n-butylammonium hydroxide  was added and incubated overnight at 100 °C (silicone oil bath) to hydrolyze all lipids. Fatty acids were esterified by adding 4 mL dichloromethane , 300 μL iodomethane , and 2 mL Milli-Q water and mixing at room temperature on a rocker for 1 h. After centrifugation at 4200 g for 10 min, the lower organic phase was washed twice with Milli-Q water, dried in fumehood overnight, and resuspended in 3 mL of diethyl ether . This was sonicated in a water-bath sonicator for 30 min for uniform resuspension of lipids. FAMES/MAMES were harvested after centrifugation and transfer of supernatant into a fresh glass vial, dried, and lipids were resuspended in 200 μL dichloromethane. Equal counts  were loaded on a silica gel 60 F254 thin-layer chromatography (TLC) plate  and resolved using hexane/ethyl acetate . To determine the distribution of FAMEs, the same sample was analyzed on a C18 reverse-phase TLC  and developed with acetonitrile/dioxane . The TLCs were visualized using a phosphorimager (GE Typhoon phosphorimager).Metabolic radiolabeling of mycolic acids and fatty acids of escribed . BrieflyMtb cultures (OD600 nm ≈ 0.6) were washed twice with ice-cold PBS and resuspended in PBS. An equal number of cells (109) for each strain were treated with [14C]-Isoniazid (ViTrax Inc) at a concentration of 0.5 μCi/mL. After 1 hr of treatment, bacteria were pelleted and extensively washed to remove residual [14C]-isoniazid. The intracellular level of isoniazid was determined by bacterial lysis using bead-beating. Lysates were added to 1,4-Bis(5-phenyl-2-oxazolyl)benzene solution in toluene , and radioactivity was measured by a liquid scintillation counter (LKB WALLAC 1209 RACKBETA liquid scintillation counter).Log-phase Mtb cells was determined as previously described (8) for each strain were added into the 96-well plate. Ethidium bromide  was added to the cells, and fluorescence intensity was measured for 1 hr using a SpectraMax M3 plate reader (Molecular Devices) in the bottom-reading mode with excitation at 530 nm and emission at 590 nm.Cell-membrane permeability of the escribed , with miMtb H37Rv strains were grown to log phase (OD600 nm = 0.6), and pyridine nucleotides (NAD+ and NADH) levels were determined by a redox-cycling assay (+ extraction) or 0.2 M NaOH . The samples were heated at 55 °C for 20 min, followed by cooling at 0 °C for 5 min. Samples were then neutralized with 0.1 M NaOH  or 0.1 M HCl . After centrifugation, the supernatant containing pyridine nucleotides was passed through a 0.2 µm filter. The redox cycling assay was performed using a reagent mixture consisting of equal volumes of 1 M bicine buffer (pH8.0) , absolute ethanol as substrate, 40 mM EDTA (pH8.0), 4.2 mM MTT , and twice the volume for 16.6 mM PES , previously incubated at 30 °C. Of the reagent mixture, 100 μL was incubated with 90 μL of cell extract followed by the addition of 10 μL alcohol dehydrogenase in 0.1 M bicine buffer, ≈ 6 U for NAD(H) estimation . The absorbance at 565 nm was recorded every minute for 10 min at 30 °C. Using 0.01–0.1 mM standard solutions of NADH  and NAD+ , a standard curve (absorbance versus time plot) was generated whose slope (∆Absorbance/ min) of the linear region was correlated to the concentration of coenzyme (mM) by a linear fit equation. This equation was used to determine the concentration(mM) of NAD/H in the samples. NADH and NAD+ concentrations were normalized to OD 600 nm.ng assay . Brieflyhttps://mycobrowser.epfl.ch/). To find similar protein sequences and structures, the BLAST program (blastp) of Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed using the query sequence of Rv0158 protein against the protein data bank (PDB). Based on the BLAST result, we selected the top hit: KstR2 protein crystal structure (PDB ID: 4W97) of TetR-family, form Mtb .Rv0158 protein sequence was taken from the Mycobrowser repository (form Mtb . This prrv0158 ORF was PCR-amplified from Mtb H37Rv genome and cloned into pet28a plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells. At OD600 nm of ∼0.6, 0.5 mM isopropyl-β-D thiogalactopyranoside  were added to induce expression of His-tagged Rv0158 for 24 hr at 18 °C. The culture medium containing the bacteria was centrifuged at 3000 g for 10 min, and the cell pellet was suspended in lysis buffer 25 mM Tris-Cl pH 8.0, 500 mM NaCl, 5% glycerol, 5 mM βmercaptoethanol, 5 mM imidazole, and 2 mM phenylmethylsulfonyl fluoride . The cells were sonicated for 30 min on ice, followed by centrifugation at 3000 g for 45 min. His-tagged Rv0158 protein in the supernatant was purified on a Ni-NTA  resin chromatography column and eluted with elution buffer 25 mM Tris-Cl pH 8.0, 500 mM NaCl, 5% glycerol, 5 mM βmercaptoethanol, 250 mM imidazole and 2 mM phenylmethylsulfonyl fluoride (PMSF), followed by dialysis to remove imidazole. The protein purity was evaluated by SDS-PAGE and quantified by BCA assay .The nucleotide sequence of For the CD spectroscopy study, the concentration of the protein sample (Rv0158) was set at 20 μM in 20 mM Tris (pH 7.5), 100 mM NaCl Buffer. Scans and melting curve experiments of the Rv0158 protein sample were performed on a JASCO J-715 spectropolarimeter. CD spectra of Rv0158 were recorded at 25 °C over the wavelength range from 260 nm to 190 nm. Prior to sample spectra, baseline spectra were obtained using 20 mM Tris (pH 7.5), 100 mM NaCl. CD melting curve was recorded at 209 nm over the temperature ranges from 10 °C to 90 °C.For gel filtration chromatography of protein sample of His-tagged Rv0158 (Ni-NTA eluted), Buffer-20 mM Tris, pH 7.5, 100 mM NaCl was used in Superdex 75 column using GE AKTA prime plus FPLC system. To find protein Rv0158 in the fractions, we performed SDS PAGE to check for bands of the expected size.10 μM of Rv0158 protein  was labelled using RED-NHS MonolithTM Protein Labeling Kit  according to manufacturer’s instructions. Each of the small molecule ligands , acetyl-CoA , propionyl-CoA , malonyl-CoA , methylmalonyl- CoA , C10-CoA , C12-CoA , C16-CoA , C18-CoA ) were titrated against the labeled protein in 1:1 dilution series starting with the highest ligand concentration of of 75 μM. A total of 16 twofold serial dilutions of the target ligand were prepared. The labeled protein was added such that the final concentration of the protein is 5 nM. Experiments were carried out using MonolithTM NT.115 MST Standard Capillaries  and measured using a Monolith NT.115 instrument with MO.Control software. Curves were fitted with a single-site binding model using PALMIST 1.5.6 software  and figurv0158 KO Mtb was transformed with ATc-inducible expression vector (pexCF-rv0158) and transformants were grown in the presence of 50 μg/mL− hygromycin B to maintain the plasmid. Log phase culture at an OD600 of 0.35 was washed twice with 1X PBS, and induced for 24 hr using a low ATc concentration 12.5 ng/mL in presence of either 0.3% (w/v) D-glucose or 0.3% (w/v) sodium propionate as sole C source. Samples for ChIP-Seq were prepared as described previously , and resuspended in IP Buffer . Cells were lysed and the supernatant was subjected to Covaris S2 ultrasonicator at settings: Duty factor = 10%, Peak power = 174 W, cycles per burst = 200, for 20 min (4 times 5 min with 1 min gap) to shear chromatin to a uniform size centred around 200 bp. Following shearing, the sample was adjusted to buffer IPP150  and shearing of DNA to an average size of 100–400 bp was confirmed by running a de-crosslinked aliquot on Agilent Bioanalyzer. Immunoprecipitation of FLAG-tagged proteins was initiated by incubating samples overnight rotating at 4 °C with Anti-FLAG M2 Magnetic Beads (Sigma Aldrich cat. no.- M8823), following manufacturer’s instructions. Samples without undergoing immunoprecipitation served as Input control for each condition. Beads were washed twice with IP buffer and once with Tris-EDTA buffer pH 7.5. Elution was performed in 50 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS for 40 min at 65 °C. All Samples were finally treated with RNAse A (1 mg/ ml) for 1 hr at 37 °C, and cross-links were reversed by incubation for 2 hr at 50 °C and for 9 hr at 65 °C in elution buffer with Protease K (10 mg/ ml). Input and Immunoprecipitated DNA were subsequently purified using QIAquick PCR purification Kit and the concentration of the DNA was measured using the Qubit HS DNA kit. NEBNExt UltraTM II DNA library Prep with sample purification beads  was used for the library preparation. Samples were sequenced on the Illumina HiSeq 2500 Sequencing System.eviously . BrieflyMycobacterium tuberculosis H37Rv, NC_000962.3) obtained from the NCBI database. Samtools (version 0.1.19-96b5f2294a) were used to map the reads with a mapping quality of at least 1 resulting in.sam file. After the conversion of.sam file to.bam, the bam files were sorted using samtools sort command. The sorted bam files of each pulldown-input pair were used to call peaks using Macs2 (version 2.2.6) callpeak function with the following parameters; --mfold 2 80 s 50 --nomodel--keep-dup all. The peaks obtained were at least 1.5- fold enriched with ≤ 0.01 Q value (FDR). To obtain the genes having overlap with the peaks, gene positions were extracted using the.gff file of the reference file (NC_000962.3), and a custom.bed format file was made. Based on the peak positions and overlap with the genes/regulatory region , genes were identified using in-house python script. A peak was associated with a gene only if both the biological replicates have at least a peak for that gene.The single-end reads with raw read quality of Phred score ≥ 20 were mapped using bwa (version 0.7.12-r1039) to the reference genome , which were further uploaded on integrative genomics viewer (IGV) software. To prepare for the line plots for the pull-down regions, bedtools coverage (version 2.30.0) was used to make coverage using a bed file made from the sorted bam file (bamtobed option) and a custom bed file of chromosome positions with 100 base pairs intervals. From the coverage file, normalised values corresponding to every bin were obtained by dividing the total number of mapped reads for every sample . Averages of both the replicates for every sample (both pull-down and input) were calculated which were further used to calculate the Fold change using the formula: (Average CPM of Pull-down samples)/(Average CPM of Input samples). Raw data has been submitted to NCBI’s Gene Expression Omnibus .For visualisation of the peaks, the sorted bam files were converted to bigwig format with the following parameters; To identify consensus motifs of Rv0158, we extracted 500 nucleotide length sequences corresponding to the peaks obtained from macs2 output. The peak summit was centred in this sequence with 250 base pairs on either side. These sequences were used as input for the MEME-ChIP program To identify the positions of the best enriched motif and known motifs  we used FIMO Mtb strains were precultured to OD 600 nm of ≈1.0, washed with 1 X PBS, and resuspended in 7H9 supplemented with either 0.3% (w/v) D-glucose or 0.3% (w/v) sodium propionate, followed by incubation at 180 RPM in a rotary shaker incubator. Log phase cells (OD = 0.8–1.2) were quenched for 5 min in 4 volumes of 60% methanol (maintained at –45 °C in dry ice–methanol bath) and then centrifuged at 4800 g (–10 °C). The pellet was re-suspended in 700 μL of 60% methanol (maintained at –45 °C in dry ice–methanol bath) and then centrifuged at 4800 g (–10 °C). The pellet was re-suspended in 1 mL of 75% ethanol and incubated at 80 °C for 3 min with intermittent vortexing at 1.5 min intervals, immediately followed by incubation on ice for 5 min and centrifugation at 17,000 g for 15 min. The supernatant was then lyophilized on a vacuum concentrator for 3–4 h and then stored at –80 °C till further analysis.Metabolite isolation and Targeted analysis of Methylcitrate and TCA cycle metabolites were done as described previously . BrieflyFor measurement of the TCA cycle and glyoxylate cycle intermediates, metabolites were derivatized with O-benzylhydroxylamine (OBHA) as mentioned previously . Oxaloac2 at 37 °C. A total of 3×105 cells/well was seeded into a 24-well cell-culture plate. RAW macrophages were activated with IFN-γ (100 U/mL)  and E. coli lipopolysaccharide  for 16 hr. The resulting activated macrophages were infected with Mtb expressing Mrx1-roGFP2 at a multiplicity of infection (MOI) of 10 and incubated for 4 hr at 37 °C in 5% CO2. After infection, extracellular bacteria were removed by washing three times with phosphate-buffered saline . For CFU determination, infected cells were lysed in 7H9 medium containing 0.06% sodium dodecyl sulfate (SDS); dilutions were prepared using 7H9 medium, and aliquots were plated on 7H11+OADC agar plates. Plates were incubated at 37 °C for 3–4 weeks before counting colonies.The murine macrophage cell line- RAW 264.7 were acquired from ATCC  and tested negative for mycoplasma contamination by DE-MycoX Mycoplasma PCR Detection Kit (CELLclone cat. no.- GX-E-250). RAW 264.7 was grown in DMEM medium (Cell Clone) supplemented with 10% heat-inactivated (55 °C) fetal bovine serum  in an incubator containing 5% COMSHE determination, infected macrophages were treated with 10 mM N-ethylmaleimide  for 5 min at room temperature, followed by fixation with 4% paraformaldehyde  for 1 hr at room temperature. Infected macrophages were analyzed using a FACS Verse Flow cytometer (BD Biosciences). Intramycobacterial MSHE was determined using the Nernst Equation as described previously  agar plates and counting visible colonies after 3–4 weeks of incubation at 37 °C.All animal studies were executed as per guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, with approval from the Institutional Animal Ethical Committee and Biosafety Level-3 Committee. 6- to 8-week-old female pathogen-free C57BL/6 J mice were infected via a low-dose aerosol exposure to the -value of less than 0.05 was considered significant. Statistical Significance was determined by unpaired two-tailed student’s t-test; one-way ANOVA was performed where a comparison of multiple groups was made.Statistical analysis was performed using GraphPad Prism version 8.4.3 software. The mean and standard error of mean values was plotted as indicated in figure legends. A pFurther information and requests for resources and reagents should be directed to the Lead Contact: Dr. Amit Singh (asingh@iisc.ac.in).This study did not generate new unique reagents. Constructs and other reagents generated in this study will be made available from the lead contact for academic/non-commercial research purposes on request without restriction under a Material Transfer Agreement. Mycobacterium tuberculosis that are required to maintain the reducing environment of the cell. Through this study, the authors provide compelling data that Rv0158 functions as a critical regulator of bacterial responses to endogenous reactive oxygen species. This study will be important to investigators interested in bacterial physiology and how this can impact pathogenesis.This manuscript is important as it describes the powerful combination of TnSeq approaches with a reporter for mycothiol redox potential to identify genes in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:Mycobacterium tuberculosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of metabolism-linked redox homeostasis in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) A more thorough analysis and dissection of Rv0158 is necessary to provide insight into its role in mycobacterial metabolism and redox homeostasis.2) The authors should be more conservative with their data interpretations and open to alternative models of Rv0158 function.Reviewer #1 (Recommendations for the authors):In this manuscript, the authors combined TnSeq approaches with a reporter for mycothiol redox potential to identify genes in Mtb that are required to maintain the reducing environment of the cell. The authors then examine the pooled library of Tn mutants that were more oxidized for evidence of DNA damage, lipid peroxidation, upregulation of oxidative stress gene expression, and inability to recover from oxidative stress. All of this validated that they had selected a pool of mutants that were on average exhibiting a more oxidized state. The authors then focus the rest of the manuscript on the transcription factor Rv0158, by generating a deletion mutant and complemented version for Rv0158 and confirming that the mutant exhibits a more oxidized condition. This is an interesting and important characterization of Rv0158 in Mtb. Based on their data, the authors propose that Rv0158 is required to calibrate the cytoplasmic redox potential in response to changes in carbon sources. However, there are multiple explanations for the phenotypes observed, and the function for Rv0158 in metabolism and redox homeostasis remains unclear.– Lines 329-331- the authors mention that docking analysis highlighted cholesterol as a potential ligand for Rv0158, but is this just because they used the KstR2 structure as a model, which would fit the Rv0158 structure to KstR2 and may not capture important differences in the ligand binding site? What is the potential model for how cholesterol sensing would allow Rv0158 to serve as a signal for redox homeostasis?– For the experiments in Figure 8, the authors state that they hypothesized that the role of Rv0158 would be carbon source dependent, but it is not immediately obvious why that is, and what the outcome for particular carbon sources would be expected. Therefore, in Lines 422-423, it is not clear that the data is in agreement with the hypothesis because it was not obvious what the hypothesis was. In particular, because the authors earlier state that Rv0158 may respond to cholesterol, why would one not expect the TF to function in the presence of cholesterol? Better flushing out of the model and mechanism would be helpful.– Figure 8 suggests that the deletion mutant is better equipped for growth on lipid carbon sources, possibly affecting metabolism and carbon source use, which impacts redox. The authors state in lines 447-448 that their findings indicate a requirement for rv0158 to calibrate the cytoplasmic redox potential in response to changes in carbon sources. However, it seems equally possible that Rv0158 has some role in carbon metabolism or assimilation of certain carbon sources into central metabolism, and this is having an indirect effect on redox potential. This may be particularly relevant since the initial experiments were performed in media with multiple available carbon sources and there was still a phenotype. The seahorse experiments also suggest issues with carbon source use.– Does the carbon source affect the sensitivity to the conditions used in 10A?– The growth defect in macrophages could just reflect the same growth defect observed in culture in glucose-containing media. It does not appear to be specific to the macrophages and this should be taken into account when interpreting that data.Reviewer #2 (Recommendations for the authors):Shee and colleagues implemented a clever TnSeq screen to identify mutants with impaired intracellular redox homeostasis. They identified, what they call the redoxosome, which includes functionally diverse genes of which 1/3rd is related to oxidative stress/antioxidant response. Network analysis led to the identification of rv0158, which encodes a putative transcriptional regulator. The authors constructed a deletion mutant and characterized it by RNAseq, seahorse measurements, lipid analyses, and during growth in a macrophage cell line and in mice. The authors conducted many experiments, which revealed that the rv0158 mutant has many phenotypes, but unfortunately the direct regulon of Rv0158 remains unidentified. The mechanistic basis for the attenuation of the rv0158 deletion mutant in the mouse model is therefore unexplained.The identified "redoxosome" includes genes that are functionally very diverse, and I am not convinced that the majority is required for redox homeostasis. Moreover, the authors failed to validate individual transposon mutants. The validation experiments done with the enriched mutant pool  are not informative as the data could be driven by just a few of the mutants. They analyzed EMSH response in a handful of transposon mutants obtained from BEI but did not carry out any further functional analysis that would provide confidence that the identified mutants are in fact required for redox homeostasis. The EMSH response is not sufficient to support this claim. It is not clear why these specific mutants were selected.The authors focused on the putative transcriptional regulator Rv0158 and used RNAseq to identify its regulon. The approach cannot distinguish genes that are directly regulated by Rv0158 from those that are indirectly regulated. This is supported by the large number of genes identified. Accordingly, the follow-up work does not lead to significant insight into the function of Rv0158. The mutant accumulated fewer mycolic acids than wt was more permeable to EtBr, and contained higher NADPH levels than wt Mtb; the underlying mechanisms and specific dysregulated genes that are responsible for these phenotypes remain undetermined. Similarly, the growth defects are difficult to interpret and the conclusion that rv0158 deletion improved growth with fatty acids as a carbon source is not supported by the data; this is only observed with propionate.in vivo growth defect and more drastic persistence defect seems inconsistent with the data from the macrophage cell line.The conclusion that rv0158 is required to survive in macrophages is incorrect. The mutant survives perfectly well ; it fails to grow like wt. The slight The MIC measurements using resazurin are a concern, given that resazurin reports on respiration, it is reduced in aerobically growing cells. The altered intracellular redox state of the rv0158 mutant could affect the resazurin reduction and confound the data.There are some inaccurate statements that need to be corrected. For example, the authors state "Mtb secretes antioxidant enzymes , superoxide dismutase (Fe-SodA), thioredoxin reductase (trxB2), Alkyl hydroperoxide reductase (AhpC) and redox buffers thioredoxin (trxC), and ergothioneine). Not all of these are secreted; in fact, the majority are not.They wrote" Mtb displays a low MIC of 3 μM for the endogenous superoxide (O2.-) generator 2, 3-Dihydro-1,4, naphthoquinone and 0.4 μM for the intracellular NO donor PA-824. On what basis do the authors quantify the MICs as "low"?The experiments shown in Figure 2 should be conducted with isolated transposon mutants.in vivo attenuation.The genes that are directly regulated by Rv0158 need to be identified to gain mechanistic insight into the many phenotypes of the mutant and potentially explain the in vivo environment to substantiate the claim that rv0158 is required for growth with fatty acids.The authors should test growth with additional fatty acids, especially those that are relevant to the The conclusion that the key rv0158 function is to maintain the flux of lipid metabolism intermediates needs to be substantiated by carbon flux analyses.MIC measurements using growth/OD600 readings should be conducted.Reviewer #3 (Recommendations for the authors):in vivo. I am concerned that the claims by the authors are not supported by the data.The authors of the study combined the Mrx-roGFP2 redox biosensor with transposon mutagenesis and FACS to identify the Mtb redoxome. From this library, the authors identify a hypothetical protein, RV0158 that modulates intracellular redox balance and lipid homeostasis. The corresponding Mtb knockout strain is implicated in anti-TB drug susceptibility and survival The strengths of the manuscript are combining roGFP with FACS to identify a set of genes involved in redox balance, implicating a regulatory gene (rv0158) involved in this process, and demonstrating its role in anti-TB drug susceptibility and virulence.The weaknesses of the manuscript include using pooled libraries of transposon mutants (the redoxome) in their Q-PCR and oxidative stress exposure experiments. Examining pooled libraries of mutant clones complicates the interpretation of data that is based on the average contribution of all the genes and makes it impossible to make reliable conclusions. Also, the redoxome was identified using a single oxidant, CHP, which is not a physiologically relevant oxidant. Hence, the redoxome is an artificial signature. An important control, an antioxidant to reverse the response, is lacking. The manuscript lacks a mechanism whereby RV0158 regulates redox homeostasis. Although the lipid metabolism, oxidative stress, and redox couple experiments provide circumstantial evidence, a clear sensing/regulatory mechanism is lacking. The authors favor that rv0158 regulates lipid metabolism as a mechanism for redox homeostasis, but unfortunately, no mechanism is provided. Lastly, I have concerns about the bioenergetic data and interpretation. Overall, numerous casually linked experiments were performed, and the manuscript appears unfocused.There are numerous grammatical issues throughout the manuscript that warrant attention.Lines 33-34; ambiguous sentences, rephrase.Lines 56-61; add references where relevant and not at the end of the sentence.Line 188; what do the authors mean by nontoxic? How was non-toxicity measured/assessed? This should be provided.Line 298-301; some of the references are outdated.Lines 313-333; this section is unnecessary and should be moved to the supplemental data.Line 423; figure 8A show delayed growth; the growth rate appears to be similar to the wild-type and complemented clones. Also, in figure 8E, the more reducing environment when cultured on fatty acid precursors compared to glucose is expected. However, only in the case of acetate (and glucose) is there a difference between the knockout and corresponding wild-type strain; propionate and cholesterol show no difference. Wise is the case and how does it relate to the role of rv0158? The authors should explain this.Lines 459-461; this data is not convincing as panels A and D are not baselined whereas all the other panels are. My suspicion is that if these data sets are baselined, there will be very little difference between the %OCR of the knockout and wild-type/complemented clones. Also, precisely how did the authors calculate the ~two-fold less/lower values?Line 457; CCCP is solely an uncoupler and does not increase carbon catabolism and it does not reveal the metabolic flexibility of Mtb; it only does so in response to particular stressors.Line 523-526; confusing sentence, rephrase.Line 1729; correctly rearrange panels E, F, and G.Line 1824; this heading is probably wrong. Respiration and not energy were measured in these experiments. Indicate that the Agilent Seahorse XFp was used to measure respiration. Also, in line 1826, extracellular acidification is not only due to glycolytic flux. This should be corrected.Line 1835; CCCP does not allow the detection of spare respiratory capacity. The authors confuse spare respiratory capacity with mammalian experiments.rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis" for further consideration by eLife. Your revised article has been evaluated by Wendy Garrett (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Biosensor-integrated transposon mutagenesis reveals The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The authors have added a tremendous amount of work to the manuscript and strengthened their dataset. They have addressed many prior concerns to yield an impressive story, but there are some concerns with the newly added data that need to be addressed. In addition, the authors still seem to bias their interpretations towards their favored model and should revise the manuscript to consider alternatives.The authors should address the following comments, which they should largely be able to do by not overstating their results, considering other models, and providing a more thorough and transparent analysis of the metabolite measurements.1. The findings that the rv0158 deletion mutant can grow on propionate at a concentration that inhibits growth of the wt and complemented strains and that Rv0158 can bind methylmalonyl-CoA are interesting. However, the authors' interpretations are often based on phenotypic differences whose physiological relevance remains unclear. Their interpretation of the phenotypes seems biased towards the model they propose, but other explanations should be considered and included, at least in the discussion.2. There are concerns regarding the metabolomics data. The authors do not seem to have used metabolite standards. Even the source data fail to show which masses they utilized for the measured metabolites. Oxaloacetate is very unstable and can typically not be measured directly. The statement "We found that α-ketoglutarate pools are marginally higher in rv0158 KO compared to WT Mtb under propionate , indicating another reason for better mutant growth on propionate" is not justified, because in the complemented mutant α-ketoglutarate pools are even higher than that in the KO.3. The authors added CFU data to assess susceptibility to antibiotics, but this is different than MIC measurements using growth as readout to assess drug resistance, which can be different than susceptibility to drug induced killing. This should be considered when describing and interpreting the data.4. The authors' explanation for the different binding targets of Rv0158 during growth in glucose and in propionate is not clear. It seems that they suggest that methylmalony -CoA binding is required for Rv1058 to bind and repress the prpR promoter, but direct evidence for this is lacking. Overall, their model of Rv0158 activity is very complex and is sometimes based solely on associations. The authors would benefit by more rigorously evaluating their data and being more careful with their interpretations. Essential revisions:1) A more thorough analysis and dissection of Rv0158 is necessary to provide insight into its role in mycobacterial metabolism and redox homeostasis.To better understand Rv0158 function in mycobacterial metabolism and redox balance, we performed several key experiments described below.rv0158 KO on glucose and mixed nutrients , whereas growth was better on fatty acids such as propionate . Therefore, to understand the better adaptability of rv0158 KO on propionate as a carbon (C) source, we performed liquid chromatography-mass spectrometry (LC-MS) metabolomics following exposure of WT Mtb and rv0158 KO to 0.3% glucose or 0.3% propionate . We assessed the pool sizes of the intermediates associated with the methylcitrate cycle (MCC), TCA, and glyoxylate cycles. Our data suggest that Rv0158 deficiency accelerates propionyl-CoA utilization and reduces the pool of toxic metabolites such as glyoxylate , thereby positively influencing the growth and redox potential of rv0158 KO utilizing propionate as a sole carbon source .One of the significant findings of our manuscript is the slow growth of Mtb. Therefore, we proposed that the physical association of methylmalonyl-CoA with Rv0158 likely facilitates the regulation of genes associated with propionate metabolism in Mtb. We performed ChIP-sequencing (ChIP-seq) of Rv0158 on glucose or propionate as a sole carbon source to examine this . On glucose, Rv0158 binds to several genes involved in methyl-branched lipid biosynthesis , lipid transporter , biotin synthesis (bioF2), and surface-associated proteins . Under propionate condition, Rv0158 binds to the genomic region encompassing 32 genes, 24 genes overlap with glucose. The promoter regions of the remaining eight genes that Rv0158 exclusively bind in propionate- grown Mtb were part of the glyoxylate cycle and MCC  .We then extended our study to understand the potential model of propionate sensing by Rv0158. We employed microscale thermophoresis (MST) to identify ligands for Rv0158 and found that it binds with malonyl-CoA, decanoyl -CoA, C12/C16/C18-CoA, and methylmalonyl-CoA. The highest affinity was for methylmalonyl-CoA (affinity constant [Kd] of 0.9 ± 0.3 µM) (Table 1 in the revised manuscript). Methylmalonyl-CoA is a crucial intermediary metabolite regulating propionate flux into MCC, methylmalonyl pathway, and methyl branched lipids in icl1, prpR, prpC, prpD) in WT Mtb, rv0158 KO, and rv0158 Comp growing on glucose or propionate by qRT-PCR. We observed increased expression of MCC genes in rv0158 KO compared to WT Mtb on glucose . Propionate exposure increased MCC gene expression in rv0158 KO compared to WT Mtb. In addition to the MCC pathway, Mtb mitigates propionate toxicity via the biosynthesis of PDIM (phthiocerol dimycocerosates). We measured the expression of the PDIM biosynthesis cluster in rv0158 KO. Expression of PDIM genes was broadly unchanged in rv0158 KO on glucose compared to WT Mtb . However, similar to MCC genes, the PDIM cluster was induced ≈10-fold in rv0158 KO compared to WT Mtb on propionate . In sum, our ChIP-seq, RT-qPCR, and MST data indicate that Rv0158 prevents the hyperexpression of MCC genes and adjusts the partitioning of propionate between the catabolism and methyl branched lipids anabolism in Mtb.Lastly, to support ChIP-seq data, we performed qRT-PCR of MCC genes (We thank the Reviewers for these excellent recommendations.2) The authors should be more conservative with their data interpretations and open to alternative models of Rv0158 function.Mtb.We have extensively revised the manuscript and provided alternative models of the Rv0158 function in Reviewer #1 (Recommendations for the authors):In this manuscript, the authors combined TnSeq approaches with a reporter for mycothiol redox potential to identify genes in Mtb that are required to maintain the reducing environment of the cell. The authors then examine the pooled library of Tn mutants that were more oxidized for evidence of DNA damage, lipid peroxidation, upregulation of oxidative stress gene expression, and inability to recover from oxidative stress. All of this validated that they had selected a pool of mutants that were on average exhibiting a more oxidized state. The authors then focus the rest of the manuscript on the transcription factor Rv0158, by generating a deletion mutant and complemented version for Rv0158 and confirming that the mutant exhibits a more oxidized condition. This is an interesting and important characterization of Rv0158 in Mtb. Based on their data, the authors propose that Rv0158 is required to calibrate the cytoplasmic redox potential in response to changes in carbon sources. However, there are multiple explanations for the phenotypes observed, and the function for Rv0158 in metabolism and redox homeostasis remains unclear.We thank the Reviewer for responding positively to our manuscript. We have addressed all of the Reviewer’s concerns in the revised manuscript.– Lines 329-331- the authors mention that docking analysis highlighted cholesterol as a potential ligand for Rv0158, but is this just because they used the KstR2 structure as a model, which would fit the Rv0158 structure to KstR2 and may not capture important differences in the ligand binding site? What is the potential model for how cholesterol sensing would allow Rv0158 to serve as a signal for redox homeostasis?in silico investigation may not provide a potential model for how Rv0158 senses fatty acids such as cholesterol and propionate for redox homeostasis. Therefore, we employed microscale thermophoresis (MST) to identify ligands for Rv0158. We tested various metabolites generated during the catabolism of long-chain fatty acids, propionate, and cholesterol (Lines: 457- 464 in the revised manuscript). We found that Rv0158 does not bind with CoA, acetyl-CoA, and propionyl-CoA. However, Rv0158 showed binding with malonyl-CoA, decanoyl -CoA, C12/C16/C18-CoA, with the highest affinity for methylmalonyl-CoA (affinity constant [Kd] of 0.9 ± 0.3 µM) (Table 2 and Table 2- Source Data 1 in the revised manuscript). Since methylmalonyl-CoA is a crucial intermediary metabolite regulating propionate flux into MCC, methylmalonyl pathway, and methyl branched lipids , we proposed that its physical association with Rv0158 likely facilitates regulation of genes associated with propionate metabolism in Mtb. We supported this model by performing ChIP- Seq and expression analysis during the growth of Mtb on glucose or propionate as a sole carbon source.We agree with the Reviewer that – For the experiments in Figure 8, the authors state that they hypothesized that the role of Rv0158 would be carbon source dependent, but it is not immediately obvious why that is, and what the outcome for particular carbon sources would be expected. Therefore, in Lines 422-423, it is not clear that the data is in agreement with the hypothesis because it was not obvious what the hypothesis was. In particular, because the authors earlier state that Rv0158 may respond to cholesterol, why would one not expect the TF to function in the presence of cholesterol? Better flushing out of the model and mechanism would be helpful.rv0158 KO. Furthermore, genes related to the methylcitrate cycle , which alleviate propionate toxicity , were significantly upregulated in rv0158 KO. On this basis, we first examined the role of rv0158 in utilizing fatty acids as nutrients by Mtb. Furthermore, fatty acids being a more reduced C substrate than glucose , we tested if differences in the growth of rv0158 KO on fatty acids vs glucose are also associated with changes in the redox potential of Mtb. Our ligand binding assay, metabolomics, ChIP-seq, and expression analysis shed new light on the mechanism of how Rv0158 could function in the presence of propionate to adjust the growth and redox potential of Mtb.We thank the Reviewer for this comment. We have simplified this section (Lines: 355- 370 in the revised manuscript). Our RNA-Seq data on media containing mixed carbon sources  indicated that genes associated with fatty acid catabolism were induced in – Figure 8 suggests that the deletion mutant is better equipped for growth on lipid carbon sources, possibly affecting metabolism and carbon source use, which impacts redox. The authors state in lines 447-448 that their findings indicate a requirement for rv0158 to calibrate the cytoplasmic redox potential in response to changes in carbon sources. However, it seems equally possible that Rv0158 has some role in carbon metabolism or assimilation of certain carbon sources into central metabolism, and this is having an indirect effect on redox potential. This may be particularly relevant since the initial experiments were performed in media with multiple available carbon sources and there was still a phenotype. The seahorse experiments also suggest issues with carbon source use.rv0158 KO utilizing propionate as a sole carbon source. We thank the Reviewer for this insightful comment.We agree with the Reviewer. Our metabolomics, ChIP-seq, and expression data confirm that Rv0158 deficiency accelerates propionyl-CoA utilization and reduces the pool of toxic metabolites such as glyoxylate, thereby positively influencing the growth and bioenergetics of – Does the carbon source affect the sensitivity to the conditions used in 10A?rv0158 KO under hydrogen peroxide (H2O2) stress when grown in the presence of acetate/propionate /cholesterol as a sole carbon source . We found that the sensitivity of rv0158 KO towards H2O2 stress was alleviated when we substituted glucose with fatty acids (0.03% acetate or 0.3% propionate or 0.02% cholesterol) as C source (Lines: 503- 508 in the revised manuscript). This aligns with the restoration of MSH redox state  and OXPHOS (relative to WT Mtb) in the knockout strain when growing with fatty acids as a C source .We thank the Reviewer for this comment. We have addressed the Reviewer’s concern by measuring the survival of – The growth defect in macrophages could just reflect the same growth defect observed in culture in glucose-containing media. It does not appear to be specific to the macrophages and this should be taken into account when interpreting that data.rv0158 KO exhibits a slow-growth phenotype in glucose-containing media. In the revised manuscript, we addressed the Reviewer’s concern by assessing if the macrophage environment induces slow growth like glucose or killing of the mutant over time (Lines: 532- 536 in the revised manuscript). Immune- activation of macrophages elevates phagosomal oxidative and nitrosative stress to cause redox stress in Mtb . Therefore, we examined the mutant phenotype inside the lipopolysaccharide (LPS), and IFN-γ activated RAW 264.7 macrophages. We observed that rv0158 KO entered bacteriostasis two days post-infection (p.i.) and showed ≈50% killing at four days p.i. . The killing of the mutant coincides with an overwhelming oxidative shift in the MSHE of Mtb inside immune-activated macrophages . At both these time points, the survival of the mutant remains significantly lower than the WT Mtb. The data agree with our findings that rv0158 KO is exceptionally sensitive to oxidative and nitrosative stress in vitro. Altogether, while glucose slows the growth of rv0158 KO in vitro, an oxidatively hostile environment of activated macrophages induces killing.We thank the Reviewer for this comment. We showed that the Reviewer #2 (Recommendations for the authors):Shee and colleagues implemented a clever TnSeq screen to identify mutants with impaired intracellular redox homeostasis. They identified, what they call the redoxosome, which includes functionally diverse genes of which 1/3rd is related to oxidative stress/antioxidant response. Network analysis led to the identification of rv0158, which encodes a putative transcriptional regulator. The authors constructed a deletion mutant and characterized it by RNAseq, seahorse measurements, lipid analyses, and during growth in a macrophage cell line and in mice. The authors conducted many experiments, which revealed that the rv0158 mutant has many phenotypes, but unfortunately the direct regulon of Rv0158 remains unidentified. The mechanistic basis for the attenuation of the rv0158 deletion mutant in the mouse model is therefore unexplained.The identified "redoxosome" includes genes that are functionally very diverse, and I am not convinced that the majority is required for redox homeostasis. Moreover, the authors failed to validate individual transposon mutants. The validation experiments done with the enriched mutant pool  are not informative as the data could be driven by just a few of the mutants. They analyzed EMSH response in a handful of transposon mutants obtained from BEI but did not carry out any further functional analysis that would provide confidence that the identified mutants are in fact required for redox homeostasis. The EMSH response is not sufficient to support this claim. It is not clear why these specific mutants were selected.The authors focused on the putative transcriptional regulator Rv0158 and used RNAseq to identify its regulon. The approach cannot distinguish genes that are directly regulated by Rv0158 from those that are indirectly regulated. This is supported by the large number of genes identified. Accordingly, the follow-up work does not lead to significant insight into the function of Rv0158. The mutant accumulated fewer mycolic acids than wt was more permeable to EtBr, and contained higher NADPH levels than wt Mtb; the underlying mechanisms and specific dysregulated genes that are responsible for these phenotypes remain undetermined. Similarly, the growth defects are difficult to interpret and the conclusion that rv0158 deletion improved growth with fatty acids as a carbon source is not supported by the data; this is only observed with propionate.in vivo growth defect and more drastic persistence defect seems inconsistent with the data from the macrophage cell line.The conclusion that rv0158 is required to survive in macrophages is incorrect. The mutant survives perfectly well ; it fails to grow like wt. The slight We thank the Reviewer for insightful comments. We have addressed most of the Reviewer’s comments in the revised manuscript. Since our FACS-based strategy did not use exogenous ROS donors  to induce oxidative stress, we expected to unbiasedly sort out mutants that directly or indirectly affect redox by influencing metabolism and respiration. Therefore, our findings are on the anticipated lines. We randomly selected 10 Tn mutants, available at the BEI resource, to validate the screen. To further bolster our validation, we analyzed the Tn mutants (ROS measurement and DNA damage) to confirm that the strains are indeed oxidatively stressed. We have included these findings in the revised manuscript .Mtb is grown in glucose vs propionate-medium. Furthermore, we performed a metabolomics study to examine the underlying mechanism of growth improvement on propionate. Finally, we examined the survival of the mutant inside immune-activated macrophages and confirmed poor survival. The mutant showed a slow growth phenotype on glucose and mixed carbon source . However, the growth of the mutant was better than WT Mtb on propionate. Overall, data suggest that Rv0158 deficiency accelerates propionyl-CoA utilization and reduces the pool of toxic metabolites such as glyoxylate, positively influencing the growth and bioenergetics of rv0158 KO utilizing propionate as a sole carbon source. We also repeated macrophages data to include more time points and showed that rv0158 KO entered into bacteriostasis at two days post-infection (p.i.) and showed ≈50% killing at four days p.i. The killing of the mutant coincides with an overwhelming oxidative shift in the MSHE of Mtb inside immune-activated macrophages. At both these time points, the survival of the mutant remains significantly lower than the WT Mtb.We selected an uncharacterized transcription factor (Rv0158) for further analysis. In the revised manuscript, we performed a detailed characterization of Rv0158. We identified the ligands for Rv1058 by MST. Using ChIP-seq, we identified binding sites for Rv0158 when Mtb persistence in mice relies upon resistance to oxidative/nitrosative stress, efficient metabolization of glucose, and co-catabolism of fatty acids and glucose . We have included this in the Discussion section of the revised manuscript.The likely reason for attenuation inside animals requires more experimentation. However, the plausible cause could be the exceptional sensitivity of the mutant to oxidative/nitrosative stress and fitness defect on diverse carbon sources or glucose as a sole carbon source. These data are consistent with the fact that The MIC measurements using resazurin are a concern, given that resazurin reports on respiration, it is reduced in aerobically growing cells. The altered intracellular redox state of the rv0158 mutant could affect the resazurin reduction and confound the data.rv0158 KO to H2O2 depends upon the type of carbon source by enumerating CFU (please refer to the response to Reviewer 1). Additionally, in the absence of antibiotics, we did not see any difference in the extent of resazurin reduction to resorufin among the untreated Mtb strains  (Lines: 512- 514 in the revised manuscript).We thank the Reviewer for this comment. We have included CFU data to corroborate the resazurin- reduction experiment in the revised manuscript . Furthermore, we confirmed that the sensitivity of There are some inaccurate statements that need to be corrected. For example, the authors state "Mtb secretes antioxidant enzymes , superoxide dismutase (Fe-SodA), thioredoxin reductase (trxB2), Alkyl hydroperoxide reductase (AhpC) and redox buffers thioredoxin (trxC), and ergothioneine). Not all of these are secreted; in fact, the majority are not.Mtb (Lines: 69- 72 in the revised manuscript).We thank the Reviewer for this comment. Based on the published literature, we have included a table (Table 1 in the revised manuscript) in the revised manuscript showing the localization of these systems in They wrote" Mtb displays a low MIC of 3 μM for the endogenous superoxide (O2.-) generator 2, 3-Dihydro-1,4, naphthoquinone and 0.4 μM for the intracellular NO donor PA-824. On what basis do the authors quantify the MICs as "low"?Mtb retains viability under high millimolar (mM) concentrations of exogenous ROS and RNI . However, it displays exceptional sensitivity to low μM (μM) concentrations of donors that permeate inside Mtb to deliver superoxide  or NO (PA-824) , . We have rephrased the sentence to make this point clearer (Lines: 92- 99 in the revised manuscript).We thank the Reviewer for this comment. Our quantification is based on the fact that The experiments shown in Figure 2 should be conducted with isolated transposon mutants.MSHE displayed by the flow-sorted Tn-mutants pool correlates with other oxidative stress markers under aerobic growth conditions. TraDIS analysis confirmed that the collection of oxidized mutants contains 368 genes with significantly greater Tn insertions than the input library. While experimentally ascribing redox function to 368 genes requires independent investigation, we begin validating our data by confirming an oxidative shift in the MSHE of strains having Tn-insertions in 10 randomly selected genes that are part of the redoxosome . We agree with the Reviewer that additional experiments are needed to show that these Tn mutants are oxidatively stressed. Therefore, we measured intracellular ROS and DNA damage in the Tn mutants to confirm that the strains are indeed oxidatively stressed leading to damage to biomolecules (DNA) (Lines: 263- 265 in the revised manuscript) . In agreement with the oxidized MSHE of Mtb mutants , higher eROS and DNA damage as compared to WT Mtb was detected.We thank the Reviewer for this comment. We performed experiments shown in Figure 2 to validate that the oxidative in vivo attenuation.The genes that are directly regulated by Rv0158 need to be identified to gain mechanistic insight into the many phenotypes of the mutant and potentially explain the rv0158 KO grew better on propionate than glucose as a sole carbon source . Furthermore, Rv0158 directly binds to methylmalonyl-CoA, a crucial intermediary metabolite regulating propionate flux into methyl citrate cycle (MCC), methylmalonyl pathway, and methyl branched lipids . Therefore, we propose that Rv0158 facilitates the regulation of genes associated with propionate metabolism in Mtb. To examine this, we performed ChIP-sequencing (ChIP-seq) using an anhydrotetracycline (ATc)-inducible flag-tagged Rv0158 (pexCF-rv0158)  (Lines: 468- 474 in the revised manuscript).We thank the Reviewer for this comment. We found that Mtb genes . Further classification of these genes indicates that Rv0158 binds to several genes involved in methyl-branched lipid biosynthesis , lipid transporter , biotin synthesis (bioF2), and surface-associated proteins  (Supplementary file 5 in the revised manuscript) (Lines: 474- 482 in the revised manuscript). Under propionate condition, Rv0158 binds to promoter regions encompassing 32 genes . Out of 32 genes, 24 genes overlap with glucose. The promoter regions of the remaining eight genes Rv0158 exclusively bound in propionate- grown Mtb were part of MCC and the glyoxylate cycle   (Lines: 483- 488 in the revised manuscript).When glucose was used as a sole C-source, Rv0158 binds to the promoter regions of 70 Mtb grown in propionate . In contrast to WT Mtb, MCC genes were uniformly induced in rv0158 KO independent of C source . However, propionate resulted in a far greater induction of MCC genes in rv0158 KO than glucose . In addition to the MCC pathway, Mtb mitigates propionate toxicity via the biosynthesis of PDIM (phthiocerol dimycocerosates)  . We measured the expression of the PDIM biosynthesis cluster in rv0158 KO. Expression of PDIM genes was broadly unchanged in rv0158 KO on glucose compared to WT Mtb . However, similar to MCC genes, the PDIM cluster was induced ≈10-fold in rv0158 KO compared to WT Mtb on propionate . Since Rv0158 did not bind to any PDIM genes, data indicate that their hyperexpression in rv0158 KO, specifically on propionate, was an indirect effect (Lines: 428- 445 in the revised manuscript).We performed RT-qPCR and found that the expression of MCC genes was specifically induced in WT Mtb cultured on glucose maintains carbon flux into MCC and glyoxylate cycle, indicating the availability of propionyl-CoA and methylmalonyl-CoA . Under propionate-rich conditions, increased requirements to mitigate toxicity can be achieved by Rv0158-mediated binding high levels of methylmalonyl-CoA and calibrating carbon flux into MCC by direct binding to prpR, prp operon, and icl1, and indirectly to production of methyl branched lipids. Lack of this control under conditions of mixed carbon sources (sugar and fatty acids) in vivo could have contributed to the persistence defect of rv0158 KO in mice. The exceptional sensitivity of the mutant to oxidative and nitrosative stress is an additional factor responsible for the phenotype of the mutant in vivo.Since Rv0158 binds many genes related to methyl-branched fatty acid metabolism/transport despite growth on glucose, we think that methylmalonyl-CoA is available for binding to Rv0158 and repressing undesirable over-expression of propionate metabolic pathways. Agreeing to this, in vivo environment to substantiate the claim that rv0158 is required for growth with fatty acids.The authors should test growth with additional fatty acids, especially those that are relevant to the Mtb, rv0158 KO, and rv0158 Comp in 7H9 medium supplemented with either 0.3% glucose or host-relevant fatty acids  as a sole carbon (C) source (Lines: 356- 361 in the revised manuscript). Both absorbance (OD 600 nm) and CFU estimation showed a slow growth phenotype with an extended lag phase on glucose . The defective growth phenotype of rv0158 KO was absent if glucose was replaced by acetate/cholesterol/palmitate . Interestingly, we observed a gradual increase in the growth of rv0158 KO on 0.3% sodium propionate, a concentration bacteriostatic to both WT Mtb and rv0158 Comp .We thank the Reviewer for this recommendation. As suggested by the Reviewer, we measured the growth of WT The conclusion that the key rv0158 function is to maintain the flux of lipid metabolism intermediates needs to be substantiated by carbon flux analyses.rv0158 KO on fatty acids, we performed liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics following exposure of WT Mtb and rv0158 KO to 0.3% glucose or 0.3% propionate (Lines: 400- 427 in the revised manuscript). We observed a ~100-fold accumulation of 2-methylcitrate (2-MC) in WT Mtb and rv0158 KO under propionate but not glucose , confirming the activation of propionate breakdown by MCC. Furthermore, downstream metabolites of propionate catabolism such as pyruvate, succinate, fumarate, malate, oxaloacetate, and glyoxylate were significantly accumulated (2-6-fold) in propionate-grown WT Mtb as compared to glucose . In contrast, these metabolites were either not accumulated  or marginally accumulated  in propionate-grown rv0158 KO , presumably due to efficient catabolism of propionate via MCC in the mutant relative to WT Mtb. We also found evidence of lesser accumulation of pyruvate and lactate in rv0158 KO . Since the pyruvate and lactate levels depend on glyoxylate shunt, MCC, and gluconeogenesis , these findings agree with the better metabolic ability of rv0158 KO to consume propionate. Conservation of α-ketoglutarate and glutamate pools via glutamine synthase (GltB/D’s) is an additional metabolic strategy to neutralize propionate toxicity in Mtb . We found that α-ketoglutarate pools are marginally higher in rv0158 KO compared to WT Mtb under propionate , indicating another reason for better mutant growth on propionate. Data suggest that rv0158 KO mitigates propionate toxicity through efficient MCC and TCA cycle metabolization.We agree with the Reviewer. Therefore, to elucidate metabolic changes associated with the better adaptability of rv0158 KO on propionate compared to glucose, support the role of Rv0158 in integrating carbon metabolism with redox balance in Mtb.We also accept that only a pulse-label of a metabolite and the estimation of label incorporation in the metabolic products can fully indicate flux, since the steady-state amounts cannot separate production from consumption. However, when observed over entire pathways, or complete sets of metabolites, they do conclusively indicate the overall metabolic state in the cell and, in many cases, correlate with overall flux. However, to be correct in our description, we only indicated pool sizes of these metabolites in the text. We hope the Reviewer would agree that these findings, together with the data showing improved growth and bioenergetics of MIC measurements using growth/OD600 readings should be conducted.rv0158 KO by >10- fold relative to WT Mtb  (Lines: 517- 519 in the revised manuscript).We have supplemented our MIC data with CFU analysis. Treatment with 1X MIC of isoniazid (MIC = 0.06 μg/mL), moxifloxacin (MIC = 0.125 μg/mL), and bedaquiline (MIC = 0.03 μg/mL) decreased the survival of Reviewer #3 (Recommendations for the authors):in vivo. I am concerned that the claims by the authors are not supported by the data.The authors of the study combined the Mrx-roGFP2 redox biosensor with transposon mutagenesis and FACS to identify the Mtb redoxome. From this library, the authors identify a hypothetical protein, RV0158 that modulates intracellular redox balance and lipid homeostasis. The corresponding Mtb knockout strain is implicated in anti-TB drug susceptibility and survival The strengths of the manuscript are combining roGFP with FACS to identify a set of genes involved in redox balance, implicating a regulatory gene (rv0158) involved in this process, and demonstrating its role in anti-TB drug susceptibility and virulence.We thank the Reviewer for this comment.The weaknesses of the manuscript include using pooled libraries of transposon mutants (the redoxome) in their Q-PCR and oxidative stress exposure experiments. Examining pooled libraries of mutant clones complicates the interpretation of data that is based on the average contribution of all the genes and makes it impossible to make reliable conclusions.MSHE of Mtb strains harboring disruption in these genes. Consistent with the findings with the pooled libraries, the MSHE of Mtb mutants displayed an oxidative shift of +10 mV to +30 mV and exhibited higher eROS and DNA damage than WT Mtb . Please see response to Reviewer 2. We propose that the set of 368 genes represents a valuable resource to understand the contribution of genetic factors in maintaining redox balance in Mtb.We thank the Reviewer for this comment. We relied on pooled transposon (Tn) mutants libraries for developing our biosensor-based strategy. Therefore, individually ascribing redox function to 368 genes redoxosome is unrealistic. However, to begin validating our data, we randomly selected 10 genes that are part of the redoxosome and analyzed Also, the redoxome was identified using a single oxidant, CHP, which is not a physiologically relevant oxidant. Hence, the redoxome is an artificial signature. An important control, an antioxidant to reverse the response, is lacking.not used CHP on Tn-library pools expressing Mrx1-roGFP2. CHP was only used on WT Mtb expressing Mrx1-roGFP2 to set the gates for oxidized bacteria in FACS. We clarified this in the Results section. In the revised ms, we mentioned that our strategy allowed gating and isolation of Tn-mutants that were basally oxidized without exposure to oxidants such as CHP (Lines: 164- 177 in the revised manuscript). Therefore, our redoxosome is not artificial but rather a signature of Mtb genes required to maintain redox balance under normal aerobic growth conditions.We thank the Reviewer for this comment. We have MSH-Eoxidized Tn mutants pool with antioxidant molecules  + thiourea (1 mM) + bipyridyl (250 μM)) significantly decreased eROS levels  (Lines: 191- 196 in the revised manuscript).As the Reviewer suggested, we have included antioxidant control in the revised manuscript. We show that the co-treatment of The manuscript lacks a mechanism whereby RV0158 regulates redox homeostasis. Although the lipid metabolism, oxidative stress, and redox couple experiments provide circumstantial evidence, a clear sensing/regulatory mechanism is lacking. The authors favor that rv0158 regulates lipid metabolism as a mechanism for redox homeostasis, but unfortunately, no mechanism is provided.. In the revised manuscript, we have performed key experiments such as ligand binding assays, ChIP-seq, qRT-PCRs, and metabolomics to understand how Rv0158 regulates growth and redox balance of Mtb in response to a carbon source .We thank the Reviewer for this commentLastly, I have concerns about the bioenergetic data and interpretation. Overall, numerous casually linked experiments were performed, and the manuscript appears unfocused.We hope that with new data, the revised manuscript alleviates several of the Reviewer’s concerns.There are numerous grammatical issues throughout the manuscript that warrant attention.We have performed extensive corrections to the manuscript to improve grammatical issues.Lines 33-34; ambiguous sentences, rephrase.We have rephrased the sentences for clarity.Lines 56-61; add references where relevant and not at the end of the sentence.Since several references support these studies, we have included a table  to match appropriate references with the relevant study.Line 188; what do the authors mean by nontoxic? How was non-toxicity measured/assessed? This should be provided.. We do not have any data to state that treatment with 10 mM of CHP for 5 min did not lead to Mtb- killing. Therefore, we have rephrased the statement- we used this treatment conditions for only inducing 100% oxidation of the biosensor in Mtb. We have cited the appropriate reference to support the findings  (Lines: 168-170 in the revised manuscript).We thank the Reviewer for this commentLine 298-301; some of the references are outdated.. We have included the recent references.We thank the Reviewer for this commentLines 313-333; this section is unnecessary and should be moved to the supplemental data.. We have reduced this section and included new data showing the binding affinity of various ligands for Rv0158.We thank the Reviewer for this commentLine 423; figure 8A show delayed growth; the growth rate appears to be similar to the wild-type and complemented clones. Also, in figure 8E, the more reducing environment when cultured on fatty acid precursors compared to glucose is expected. However, only in the case of acetate (and glucose) is there a difference between the knockout and corresponding wild-type strain; propionate and cholesterol show no difference. Wise is the case and how does it relate to the role of rv0158? The authors should explain this.. The Reviewer is correct. Absorbance (OD 600 nm) and CFU estimation showed a slow growth phenotype with an extended lag phase on glucose . The defective growth phenotype of rv0158 KO was absent if glucose was replaced by acetate/cholesterol/palmitate . Since fatty acids restored the growth of rv0158 KO to WT Mtb levels, our findings showing comparable MSHE of rv0158 KO is likely. We only see the marginal difference in acetate, which did not restore in rv0158 Comp. We have explained why the mutant shows redox imbalance on glucose (Lines: 641- 653 in the revised manuscript).We thank the Reviewer for this commentprpR, prpC, prpD, icl1, and PDIM genes in rv0158 KO on glucose could have directed a greater flux of glucose carbon away from energy-generating TCA cycle/oxidative phosphorylation to MCC/glyoxylate cycle and energy-requiring polyketide lipid anabolism. The expression of the MCC pathway is activated by a transcription factor PrpR in Mtb, whose activity depends on an oxidation-resistant 4Fe-4S cluster . Furthermore, PrpD retained activity under high ROS conditions in other bacteria . Therefore, it is likely that MCC enzymes remained functionally active in rv0158 KO on glucose despite higher eROS. These metabolic changes are possible reasons behind altered redox balance, respiration slow down, and impaired growth of rv0158 KO on glucose.We found that Rv0158 is a repressor of the methyl citrate cycle (MCC) and the glyoxylate cycle. The constitutive hyperexpression of Lines 459-461; this data is not convincing as panels A and D are not baselined whereas all the other panels are. My suspicion is that if these data sets are baselined, there will be very little difference between the %OCR of the knockout and wild-type/complemented clones. Also, precisely how did the authors calculate the ~two-fold less/lower values?. We have replotted the %ECAR and %OCR in Mtb strains growing over glucose. All the data points are baselined to the third reading obtained just before the addition of C source . The approximate 2-fold change is determined by comparing the third reading after C- source addition and the first reading after CCCP addition, across groups (Lines: 2010-2014 in the revised manuscript).We thank the Reviewer for this commentLine 457; CCCP is solely an uncoupler and does not increase carbon catabolism and it does not reveal the metabolic flexibility of Mtb; it only does so in response to particular stressors.We have clarified this point in the revised manuscript (Lines: 386- 387 in the revised manuscript). We thank the Reviewer for pointing this out.Line 523-526; confusing sentence, rephrase.. We have removed the confusing statement.We thank the Reviewer for this commentLine 1729; correctly rearrange panels E, F, and G.. This has been corrected.We thank the Reviewer for this commentLine 1824; this heading is probably wrong. Respiration and not energy were measured in these experiments. Indicate that the Agilent Seahorse XFp was used to measure respiration. Also, in line 1826, extracellular acidification is not only due to glycolytic flux. This should be corrected.We have corrected these issues (Lines: 1997- 1998 in the revised manuscript). We thank the Reviewer for pointing this out.Line 1835; CCCP does not allow the detection of spare respiratory capacity. The authors confuse spare respiratory capacity with mammalian experiments.We thank the Reviewer for this comment. We have corrected this issue in the manuscript (Lines: 2006- 2009 in the revised manuscript).References:Mycobacterium tuberculosis using transgenic mice. Tubercle and Lung Disease 78:237–246. doi:10.1016/S0962-8479(97)90004-6Adams LB, Dinauer MC, Morgenstern DE, Krahenbuhl JL. 1997. Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to Mycobacterium tuberculosis during Infection. PLoS Pathogens 10. doi:10.1371/journal.ppat.1003902Bhaskar A, Chawla M, Mehta M, Parikh P, Chandra P, Bhave D, Kumar D, Carroll KS, Singh A. 2014. Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis. Molecular Microbiology 48:453–464. doi:10.1046/j.1365-2958.2003.03438.xBraunstein M, Espinosa BJ, Chan J, Belisle JT, Jacobs WR. 2003. SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Vibrio cholerae Encode a New Fe/S-Dependent 2-Methylcitrate Dehydratase Enzyme That Requires prpF Function in vivo. Journal of Bacteriology 186:454–462. doi:10.1128/JB.186.2.454-462.2004Grimek TL, Escalante-Semerena JC. 2004. The acnD Genes of Shewenella oneidensis and Journal of Bacteriology 200. doi:10.1128/JB.00137-18Ke N, Landeta C, Wang X, Boyd D, Eser M, Beckwith J. 2018. Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation. Mycobacterium tuberculosis Persistence in Mice. PLoS Pathogens 9. doi:10.1371/journal.ppat.1003116Marrero J, Trujillo C, Rhee KY, Ehrt S. 2013. Glucose Phosphorylation Is Required for Mycobacterium tuberculosis after acquisition of Isoniazid resistance: Individual nature of katG mutants and the possible role of AhpC. PLoS ONE 11:e0166807. doi:10.1371/journal.pone.0166807Nieto LMR, Mehaffy C, Creissen E, Troudt JL, Troy A, Bielefeldt-Ohmann H, Burgos M, Izzo A, Dobos KM. 2016. Virulence of Mycobacterium tuberculosis. Journal of Biological Chemistry 291:7060–7069. doi:10.1074/jbc.M115.707430Noy T, Vergnolle O, Hartman TE, Rhee KY, Jacobs WR, Berney M, Blanchard JS. 2016. Central role of pyruvate kinase in carbon co-catabolism of Antimicrobial Agents and Chemotherapy 57:3202–3207. doi:10.1128/AAC.02572-12Sao Emani C, Williams MJ, Wiid IJ, Hiten NF, Viljoen AJ, Pietersen R-DD, van Helden PD, Baker B. 2013. Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis. Mycobacterium tuberculosis : Implications for Propionate Metabolism during Growth on Fatty Acids. Journal of Bacteriology 190:3886–3895. doi:10.1128/JB.01767-07Savvi S, Warner DF, Kana BD, McKinney JD, Mizrahi V, Dawes SS. 2008. Functional Characterization of a Vitamin B 12 -Dependent Methylmalonyl Pathway in Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species. Antimicrobial Agents and Chemotherapy 66. doi:10.1128/aac.00592-22Shee S, Singh S, Tripathi A, Thakur C, Kumar AT, Das M, Yadav V, Kohli S, Rajmani RS, Chandra N, Chakrapani H, Drlica K, Singh A. 2022. Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis culture filtrate. PLoS ONE 15:e0221837. doi:10.1371/journal.pone.0221837Tucci P, Portela M, Chetto CR, González-Sapienza G, Marín M. 2020. Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis CPT31 reveals increased production of EsxB and proteins involved in adaptation to intracellular lifestyle. Pathogens and Disease 74:ftv127. doi:10.1093/femspd/ftv127Vargas-Romero F, Guitierrez-Najera N, Mendoza-Hernández G, Ortega-Bernal D, Hernández-Pando R, Castañón-Arreola M. 2016. Secretome profile analysis of hypervirulent Mycobacterium tuberculosis growth in macrophages. Scientific Reports 8:1–12. doi:10.1038/s41598-017-18547-9Wong D, Li W, Chao JD, Zhou P, Narula G, Tsui C, Ko M, Xie J, Martinez-Frailes C, Av-Gay Y. 2018. Protein tyrosine kinase, PtkA, is required for The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The authors have added a tremendous amount of work to the manuscript and strengthened their dataset. They have addressed many prior concerns to yield an impressive story, but there are some concerns with the newly added data that need to be addressed. In addition, the authors still seem to bias their interpretations towards their favored model and should revise the manuscript to consider alternatives.The authors should address the following comments, which they should largely be able to do by not overstating their results, considering other models, and providing a more thorough and transparent analysis of the metabolite measurements.1. The findings that the rv0158 deletion mutant can grow on propionate at a concentration that inhibits growth of the wt and complemented strains and that Rv0158 can bind methylmalonyl-CoA are interesting. However, the authors' interpretations are often based on phenotypic differences whose physiological relevance remains unclear. Their interpretation of the phenotypes seems biased towards the model they propose, but other explanations should be considered and included, at least in the discussion.Mtb and how this association affects direct interaction with DNA and gene expression. Also, the lack of Rv0158 may induce a physiological state in Mtb that is more tolerant to toxicity caused by fatty acids such as propionate. For example, cAMP homeostasis is associated with modulating redox balance, respiration, fatty acid uptake, and fatty acid toxicity in mycobacteria . Moreover, Mycobacterium bovis BCG elevates cAMP levels in response to propionate, and phosphodiesterases such as Rv0805 regulate cAMP homeostasis to modulate growth on propionate . Other evidence suggests that reduced cAMP levels result in overactive fatty acid catabolism  as we observed in the case of rv0158 KO growing on propionate. While overactive fatty acid catabolism is generally detrimental due to the accumulation of toxic intermediates, mycobacteria have evolved sophisticated regulatory mechanisms  to direct the flux of excess fatty acids away from catabolism to lipid anabolism . These alterations on the mycobacterial surface might be important to counteract toxicity caused by fatty acid-dependent disruption of the Mtb envelope . We observed a higher expression of the PDIM biosynthesis cluster, altered mycolic acids content, and changes in membrane permeability of rv0158 KO, suggesting overall changes in the cell envelope of the mutant. Therefore, the physiological effects of rv0158 disruption in Mtb could result from multiple effectors such as redox, respiration, and cAMP. Also, propionate metabolism is regulated by multiple transcription factors  in Mtb . Therefore, future experiments are needed to understand the breadth of Rv0158-mediated gene regulation in collaboration with additional regulators and metabolites  in coordinating fatty acid metabolism. In the revised manuscript, we have included the above explanations in the Discussion section of the revised manuscript (Lines: 737- 767 in the revised manuscript). We thank the editors for these insightful comments.We agree with the comment. Our data suggest that Rv0158 likely senses methylmalonyl-CoA and regulates the expression of pathways associated with propionate metabolism. However, several issues remain to be addressed. We need to investigate if methylmalonyl-CoA is perceived as a metabolic signal by Rv0158 in 2. There are concerns regarding the metabolomics data. The authors do not seem to have used metabolite standards. Even the source data fail to show which masses they utilized for the measured metabolites. Oxaloacetate is very unstable and can typically not be measured directly.We apologize for not adequately describing the metabolomics data. For measurement of the TCA cycle and glyoxylate cycle intermediates, metabolites were derivatized with O-benzylhydroxylamine (OBHA) after extraction and drying down, as mentioned in earlier method papers . In particular, oxaloacetate is unstable in aqueous solutions, hence was stabilized by OBHA derivatization to prevent degradation  . We have used metabolites standards to identify and quantify the TCA and methylcitrate cycle metabolites . The metabolites were separated using Synergi 4µm Fusion-RP 80 Å LC column  on Shimadzu Nexera UHPLC system. Solvents used for the separation of TCA intermediates are the following: 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). The metabolites were detected using ABSciex QTRAP 5500 mass spectrometer. The detailed parameters used are described in Walvekar et al., 2018. The data was acquired using Analyst 1.6.2 software (Sciex) and analyzed using MultiQuant version 3.0.1 (Sciex). We have incorporated this information in the results (Lines: 412- 419 in the revised manuscript) and methods sections (Lines: 1344- 1355 in the revised manuscript) of the revised manuscript.The statement "We found that α-ketoglutarate pools are marginally higher in rv0158 KO compared to WT Mtb under propionate , indicating another reason for better mutant growth on propionate" is not justified, because in the complemented mutant α-ketoglutarate pools are even higher than that in the KO.eLife-80218R1) but did not delete it from the corresponding response letter. This result is not a part of the revised manuscript. We apologize for the confusion.This was an inadvertent error from our side. We removed the statement from the submitted revised manuscript . However, in light of the above comment, we have modified the Results section to clearly differentiate between resistance and drug induced killing (Lines- 528- 539 in the revised manuscript).4. The authors' explanation for the different binding targets of Rv0158 during growth in glucose and in propionate is not clear. It seems that they suggest that methylmalony -CoA binding is required for Rv1058 to bind and repress the prpR promoter, but direct evidence for this is lacking. Overall, their model of Rv0158 activity is very complex and is sometimes based solely on associations. The authors would benefit by more rigorously evaluating their data and being more careful with their interpretations.-109) . While the binding motif is unique, it is closely located to the binding motifs of other transcription factors involved in regulating methylcitrate cycle  (Supplementary file 6 in the revised manuscript), suggesting overlapping or competing mechanisms to regulate propionate metabolism in Mtb. We also made it clear that the direct or indirect nature of Rv0158-mdiated regulation needs future experimentation (Lines- 508- 515 in the revised manuscript).We agree that the model of Rv0158 mediated gene regulation is very complex and needs independent investigation. To the extent possible, we have rigorously analysed and interpreted the data. We reanalyzed the ChIP-seq data and made further revision in our interpretations. 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