A LexA-independent redox-based transcriptional regulation to DNA Damage Response System in Mycobacteria

A LexA-independent redox-based transcriptional regulation to DNA Damage Response System in Mycobacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A LexA-independent redox-based transcriptional regulation to DNA Damage Response System in Mycobacteria Sige Li, Zhiyong Wu, Wei Zhou, Chun Wang, Xindi Huang, Wenjing Yu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9338042/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: The bacterial DNA damage response (DDR) system promotes survival under stressful conditions and facilitates the emergence of drug-resistant mutations. The DDR system in Mycobacterium tuberculosis is primarily governed by LexA-mediated transcriptional repression, yet whether additional regulatory inputs contribute to DDR remains to be characterized. Results: By performing a bacterial one-hybrid screen, we identified several transcriptional regulators, including Rv2282c, Rv1985c and Rv1990c, that activate DDR gene expression and inhibit cell growth. Promoter truncation analysis revealed that these regulators act through sequences distinct from the canonical LexA-binding motif. No specific interaction between these regulators and DDR gene promoters was detected by EMSAs, indicating an indirect regulatory mechanism. RNA-seq analysis revealed that approximately 25% genes were differentially expressed upon overexpression of each of these regulators. Among these, tpx , which is responsible for oxidative stress defense, was consistently repressed by all of the three DDR-activating regulators but not by control regulators. Complementation of Tpx alleviated ROS accumulation and restored cell growth in strains overexpressing these regulators, suggesting that the DDR activation is linked to redox imbalance caused by Tpx repression. Conclusions: Our data demonstrate that Rv2282c, Rv1985c, and Rv1990c activate the DDR through perturbation of redox homeostasis, revealing a previously unrecognized, LexA-independent regulation pathway in mycobacteria. Mycobacteria DNA damage response Mutasome ROS Transcriptional regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Mycobacterium tuberculosis ( Mtb ) represents one of the most successful pathogens, owing to its capability to persist within host macrophages and establish chronic infection [ 1 , 2 ]. With the continuous emergence of multidrug-resistant and extensively drug-resistant strains over recent decades [ 3 – 5 ], the treatment of Mtb infection has become increasingly challenging. Host macrophages impose a variety of stresses, including oxidative stress, to eliminate infection and prevent persistent infection [ 6 ]. Reactive oxygen species (ROS) generated by NADPH oxidase and cathepsins in macrophages, contribute to Mtb clearance [ 7 ]. In addition, certain first-line anti-tuberculosis drugs and reagents can exert bactericidal effects through their ability to induce oxidative stress in mycobacterial species and cause genomic damages [ 8 – 10 ]. DNA repair is intimately coupled with the regulation of cell division in bacteria. For example, in Escherichia coli , SulA inhibits cell division to promote DNA repair [ 11 ]. In Bacillus subtilis , the checkpoint protein YneA prevents cell division upon DNA damage [ 12 ]. In response to DNA damage, Mtb has evolved distinct DNA damage response (DDR) systems —such as the SOS system and the PafBC system—governed by specific transcriptional regulators to survive genotoxic stress. The SOS system is regulated by the transcriptional repressor LexA [ 13 ], whereas the PafBC system is activated by the transcriptional activator complex PafBC [ 14 ], which directly induces gene expression upon DNA damage. In mycobacteria, the SOS and PafBC regulons both contain the checkpoint protein ChiZ, which modulates DDR and cell division to promote survival under stress conditions[ 15 , 16 ], suggesting a conserved relationship between cell division and DDR. DDR involves translesion DNA synthesis, which is performed by the mutasome complex, comprising the C-family DNA polymerase DnaE2 and its accessory factors ImuA and ImuB [ 17 ]. The low fidelity of the mutasome promotes survival under genotoxic stress and facilitates the emergence of drug resistance [ 18 ]. As a core component of the DDR system, the mutasome is directly regulated by LexA, and is also influenced by PafBC [ 19 ]. Previous studies have indicated the existence of additional, as-yet-uncharacterized mechanisms regulating DDR-related genes, as evidenced by the observation that Mtb retains viability under mitomycin C stress even when both the SOS and PafBC systems are disrupted [ 20 ]. These findings further suggest that other transcriptional regulators are involved in DDR modulation through uncharacterized mechanisms [ 21 ]. In this study, we aimed to identify and characterize the transcriptional regulators involved in regulating the DDR systems in mycobacteria. Using a bacterial one-hybrid screening system [ 21 ], we revealed that Rv2282c, Rv1985c and Rv1990c activate the DDR system and inhibit cell growth through a LexA-independent pathway. These regulators drive widespread perturbation of gene expression and induce oxidative stress by regulating Tpx expression. Our data suggest that Rv2282c, Rv1985c and Rv1990c modulate cell division and DDR by regulating oxidative stress responses, revealing a previously unrecognized regulation to DDR independent of the canonical LexA regulatory pathway. Methods Bacterial strains, plasmids and culture conditions M. smegmatis mc 2 155 ( Ms ) was cultured in Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80, 0.5% glycerol and 0.5% dextrose or on Middlebrook 7H10 agar plates supplemented with 0.5% glycerol at 37 ℃. E. coli strains were cultured in Luria-Bertani (LB) broth or on LB agar plates at 37 ℃. The pMV306 were used for expressing proteins in mycobacteria [ 22 ]. The pZT100 plasmid was used for the construction of the reporter system [ 23 ], and pUV15TetORm was used for the construction of overexpression plasmids [ 24 ]. The following antibiotics were used at the indicated concentrations: kanamycin (BiBi), 20 µg/mL; anhydrotetracycline (ATc; American chemistry), 50 ng/mL; streptomycin, 20 µg/mL on Middlebrook 7H10 agar or 10 µg/mL on 7H9 medium; hygromycin B, 50 µg/mL. Gene deletions were constructed by homologous recombination. Mutant strains were confirmed by PCR using a pair of primers located outside the cloned region, followed by sequencing of the amplified PCR products. Plasmids and primers utilized in this study were generated and listed in Supplementary Table 4. RNA extraction and RNA-seq analysis For RNA isolation, the Ms strains carrying the expression plasmids were grown in 7H9 broth supplemented with 50 ng/mL ATc and induced at 37 ℃ for 6 h. Cells were then collected and the total RNA was extracted from bacterial cell pellets by liquid nitrogen grinding, followed by extraction using TRIzol reagent (Invitrogen, USA) as described previously [ 25 ]. For RNA-seq, rRNA was depleted using the Ribo-off RNA Depletion Kit (Vazyme). RNA libraries were constructed with VAHTS Universal RNA-seq Library Prep Kit for Illumina (Vazyme). Sequencing was conducted on the Illumina HiSeq X Ten platform using 2x150bp paired-end reads. Raw reads were quality-filtered using fastp [ 26 ]. rRNA reads were removed by Bowtie2 alignment against rRNA references [ 27 ]. Clean reads were aligned to genome of Ms (NC_008596) using STAR [ 28 ], and gene counts were generated with featureCounts [ 29 ]. Differential expression analysis was conducted using DESeq2 [ 30 ], with significance defined as fold change ≥ 2 and adjusted P value ≤ 0.05. Quantitative real-time PCR assays Removal of contaminating genomic DNA and reverse transcription were performed using the HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Vazyme, R333-01, China). The process of qRT-PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad, USA). Primers specific to target genes were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Gene-specific primers used are listed in Supplementary Table 4. Expression levels were normalized to sigA mRNA as the endogenous control. Data were obtained from three independent biological replicates, each measured in technical duplicate, and analyzed using CFX Manager (Bio-Rad) software. Bacterial one-hybrid assay The imuAB and dnaE2 promoters were fused to the promoter-less lacZ gene in pZT100 plasmid and transformed into E. coli K-12 Δ lacZ strain to generate the reporter strains K-12 imuAB p- lacZ and dnaE2 p- lacZ respectively. The coding regions of 185 transcriptional regulators derived from Mtb were fused to the rpoA gene in pRAT103 plasmid and then transformed into the K-12 imuAB p- lacZ and dnaE2 p- lacZ strains respectively. Strains were then cultured at 30 ℃ to an OD 600 of ~ 0.8 to test the activity of β-galactosidase as described previously [ 21 ]. Data were obtained from three independent biological replicates, each assayed in technical duplicate. Promoter activity analysis in mycobacteria Promoter activity in mycobacteria was analyzed as described previously [ 13 ]. Promoters were amplified from Mtb H37Rv genomic DNA using the specific primers (Supplementary Table 4) and were fused to a promoter-less mCherry gene on the pMV306 plasmid. Recombinant plasmids were co-electroporated with the transcriptional regulator-expressing plasmid pUV15TetORm into Ms wild-type strain. Cells were cultured at 37 ℃ in 7H9 broth to an OD 600 of ~ 0.3, followed by the incubation with 50 ng/mL ATc. Cells were sampled, and promoter activity was quantified as relative fluorescence units (RFU; fluorescence intensities per OD₆₀₀ unit) using a BioTek Synergy H1 microplate reader. All assays were performed in three independent experiments. Growth assay of the strains expressing the transcriptional regulator Transcriptional regulators were amplified from Mtb H37Rv genomic DNA using the specific primers (Supplementary Table 4) and cloned into pUV15TetORm. Recombinant plasmids were electroporated into Ms strain. Cells were cultured to mid-logarithmic phase in 7H9 broth at 37 ℃, followed by the incubation with 50 ng/mL ATc. Cells were sampled every 3 h, and optical density at 600 nm (OD₆₀₀) was measured using a BioTek Synergy H1 microplate reader. All assays were performed in three independent experiments. For growth on agar plates, cells were grown to an OD 600 of ~ 0.4, serially 10-fold diluted, and spotted onto agar plates supplemented with hygromycin B and ATc. Plates without ATc were served as a negative control. All plates were cultured at 37 ℃ for 3 days and imaged using an Epson imaging system. Assays were performed in two independent experiments. Measurement of reactive oxygen species Intracellular ROS levels were quantified using a Reactive Oxygen Species Assay Kit (Beyotime, China) [ 31 ]. Ms strains carrying pUV15TetORm plasmid were cultured at OD600 of ~ 0.3, and ATc was then added to a final concentration of 50 ng/mL to induce for approximately 6 h. Rosup (Beyotime, China) was added at the dilution of 1:1000 as positive control to induce the ROS. Cells were then collected and pellets were washed and centrifuged three times in 7H9 broth with 50 µg/mL hygromycin. Cells were then resuspended in 7H9 medium with 50 µg/mL hygromycin, and DCFH-DA was added to each sample at a 1:1000 dilution. Cultures were then incubated at 37 ℃ away from light for approximately 0.5-1 h. 7H9 broth containing 50 µg/mL hygromycin was then used to wash cells at least three times to remove residual dye. Cultures were finally resuspended and fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Serial dilutions of cells were plated on agar plates and incubated at 37 ℃ for 3 d. Colonies were counted to determine viable cell counts. Assays were performed in three independent experiments. Protein expression and purification Transcriptional regulator genes were amplified from Mtb H37Rv genomic DNA using the specific primers (Supplementary Table 4) and cloned into pET21a. For protein expression, recombinant plasmids were transformed into E. coli BL21 (DE3), and strains were grown at 37 ℃ to an OD 600 of ~ 0.6. Expression was induced with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 ℃ for 20 h [ 25 ]. Cell pellets were collected and resuspended lysis buffer (40 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.9). Suspensions were disrupted by sonication and clarified by high-speed centrifugation. The supernatant was collected and mixed with a prepacked His column, and non-specifically bound proteins were removed by washing with wash buffer (40 mM Tris, 300 mM NaCl, 10% glycine and 50 mM imidazole (pH 7.9). Target proteins were eluted with elution buffer (40 mM Tris, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 7.9). Eluted fractions were collected and analyzed by SDS-PAGE. DNA-binding analysis EMSA was performed as described previously [ 21 ]. Briefly, 200–300 bp FAM-labeled promoter fragments were amplified by PCR and then purified using a gel extraction kit (Omega). Promoter fragments (30 nM) were incubated with purified transcriptional regulator proteins in TB buffer (20 mM Tris - HCl, pH 7.9, 50 mM NaCl, 5 mM MgSO 4 , 1 mM dithiothreitol, 0.1 mM EDTA, 5% glycerol) at 37°C for 15–30 min. Samples were resolved on a 6% native 0.5 × TBE-PAGE gel at 100 V for 1 h. Gels were scanned using an Amersham Typhoon imager (GE Healthcare). Statistical analysis The p values shown were calculated using two-tailed Student’s t test: no significance (ns), p > 0.05; * , p ⩽ 0.05; ** , p ⩽ 0.01; *** , p ⩽ 0.001. Results Screening transcriptional regulators of DDR system To identify transcriptional regulators that regulate the DDR system, we performed an in vitro large-scale bacterial one-hybrid screen using a library of 185 transcriptional regulators from Mtb [ 21 ]. As ImuAB and DnaE2 function as the mutasome complex in the DDR (Fig. 1 A), we used both imuAB and dnaE2 promoters derived from Mtb H37Rv as targets for regulator screening. Their SOS-box sequences were mutated to abolish LexA-mediated regulation and were named as imuABp-sosmt and dnaE2p-sosmt respectively (Fig. 1 B). Each promoter was transcriptionally fused to a lacZ reporter gene, and the regulatory activity was quantified by measuring the β-galactosidase activity in E. coli ( Fig. 1 B ) . The Bacterial one-hybrid screening revealed 69 transcription factors capable of regulating the SOS-box-mutated mutasome promoters (Fig. 1 C, Supplementary Table 1). Certain regulators are indicated to be involved in regulating imuABp - sosmt and dnaE2p - sosmt (Fig. 1 C, D). As ImuAB functions together with DnaE2 in the DDR, we next examined whether these regulators would regulate both promoters using a mycobacterial fluorescent reporter system. Briefly, imuABp-sosmt or dnaE2p-sosmt was fused to a mCherry fluorescence gene, and each of the four candidate regulators was expressed in Ms under anhydrotetracycline (ATc) induction (Fig. 1 E). Interestingly, overexpression of Rv2282c, Rv0195, Rv1985c, Rv1776c, or Rv1990c in Ms activated both imuABp-sosmt and dnaE2p-sosmt (Fig. 1 F, G). Furthermore, we observed that overexpression of these regulators uniformly inhibited cell growth both in liquid medium and on agar plates (Supplementary Fig. 1A, B). These data suggest that Rv2282c, Rv0195, Rv1985c, Rv1776c and Rv1990c can activate the mutasome complex from DDR system and are associated with growth inhibition in mycobacteria. Rv2282c, Rv0195, Rv1985c, Rv1776c and Rv1990c regulate mutasome in a LexA-independent manner To characterize the regulatory mechanisms of the identified transcriptional activators, we performed a promoter mutation and truncation analysis (Fig. 2 A). For the imuAB promoter, we first tested whether the imuABp-sosmt promoter was LexA-independent. Compared with the wild-type promoter, mutation of the SOS-box motif increased the promoter activity by over 10-fold and abolished LexA-mediated repression (Fig. 2 B), confirming the SOS-box mutation in imuABp-sosmt promoter effectively eliminated LexA regulation. We then truncated the imuABp-sosmt promoter (Fig. 2 A), and found that Rv2282c Rv0195 and Rv1985c still activated imuABp-sosmt2 but had no effect on imuABp-sosmt3 (Fig. 2 C), suggesting that the region from − 138 to -88 upstream of the transcriptional start site (TSS) is critical for their regulation. In contrast, Rv1776c did not activate imuABp-sosmt2 (Fig. 2 C), implying its regulatory effect is distinct from other regulators. Rv1990c retained regulatory activity toward imuABp-sosmt3 (Fig. 2 C), suggesting that its recognition site lies proximal to the SOS-box. For the dnaE2 promoter, we also performed promoter truncation and mutation analysis (Fig. 2 D). As seen in Fig. 2 E, LexA similarly failed to repress the SOS-box-mutated dnaE2p-sosmt promoter. Rv2282c, Rv0195, Rv1990c and Rv1776c lost their regulatory activity when the promoter was truncated to dnaE2p-sosmt2 (Fig. 2 F), thereby their recognition sites are localized in the region from − 192 to -136 upstream of the TSS. Rv1985c activated dnaE2p-sosmt2 , its recognition site lies in the region − 136 to -82 upstream of the TSS (Fig. 2 F). Together, we conclude that, although these regulators activate the mutasome promoters through different DNA regions, they all act in a LexA-independent pathway, as their regulatory activity requires a DNA region upstream of promoter, and mutation of the SOS-box did not influence the regulatory effects. Non-specific binding to mutasome promoters by Rv2282c, Rv1990c and Rv1985c To investigate whether these regulators directly bind to the mutasome promoters, we performed electrophoretic mobility shift assay (EMSA). Rv2282c, Rv1990c and Rv1985c were successfully purified (Supplementary Fig. 2), whereas Rv0195 could not be obtained despite multiple attempts under various purification conditions. As these three regulators regulated both imuABp-sosmt2 and dnaE2p-sosmt , we select these two promoters for EMSA analysis. An unrelated promoter cas6 p [ 32 ] was selected as a negative control. Rv1985c and Rv1990c displayed binding affinity to the mutasome promoters and to cas6 p with comparable affinity (Fig. 3 A, B), indicating that these interactions are likely nonspecific. Rv2282c showed no detectable binding to either mutasome promoter or cas6 p (Fig. 3 C), suggesting that Rv2282c may not directly bind mutasome promoter DNA. In conclusion, these data indicate that Rv2282c, Rv1990c and Rv1985c may not specifically bind to mutasome promoters and may regulate DDR system through indirect way. Regulatory targets of Rv2282c, Rv1985c and Rv1990c To identify the downstream targets of these transcriptional regulators, we conducted the RNA-seq analysis in Ms strains overexpressing either Rv2282c, Rv1985c or Rv1990c. Induction of each regulator altered the expression of ~ 25% of all genes compared with the strain carrying the empty vector control (Supplementary Table 2). Differentially expressed genes were grouped into five clusters based on their expression patterns (Fig. 4 A). Genes associated with DNA replication and damage response were mainly distributed in cluster 3, and were markedly activated by all of these three regulators (Fig. 4 B; Supplementary Fig. 3). In contrast, genes involved in cell division were mainly assigned to cluster 5 and were greatly repressed (Fig. 4 C). Oxidative stress-related genes (Fig. 4 D), such as the trx-trxB operon, which encodes a thioredoxin system involved in redox balance [ 33 ], were upregulated, indicating that Rv2282c, Rv1985c and Rv1990c may perturb the redox homeostasis of mycobacteria. We next performed quantitative real-time PCR (qRT-PCR) assays to validate the RNA-seq results. Each transcript was normalized to the expression level of an essential RNA polymerase sigma factor, sigA . Consistent with the RNA-seq data, genes associated with DDR and oxidative stress showed enhanced expression compared to the empty vector control (Fig. 4 E). As these regulators all inhibited mycobacterial growth, we next examined whether DDR regulation was mediated by growth arrest. We therefore selected Rv0047c, Rv3574 and Rv0602c, which also inhibited the growth of Ms for further RNA-seq analysis (Supplementary Fig. 4). Genes involved cell division were also repressed by these regulators, but genes involved in oxidative stress were differentially regulated by the DDR-activating regulators (Rv2282c, Rv1985c and Rv1990c), but not by Rv0047c, Rv3574 or Rv0602c (Fig. 4 D; Fig. 4 F; Supplementary Table 3). Together, our data suggest that Rv2282c, Rv1985c and Rv1990c activate of the DDR by reprogramming of redox-associated gene expression. Tpx relieves oxidative stress and growth inhibition induced by Rv2282c, Rv1985c and Rv1990c RNA-seq analysis revealed that oxidative stress response may be involved in DDR regulation. Notably, the thiol peroxidase Tpx (encoded by MSMEG_3479 ), which is involved in ROS detoxification, was downregulated by Rv2282c, Rv1985c and Rv1990c but not by the control regulators Rv0047c, Rv3574 and Rv0602c (Fig. 4 G; Fig. 5 A). A previous study showed that Tpx is an efficient peroxidase to eliminate ROS in bacteria [ 33 ], indicating that reduced Tpx expression may be associated with the increased oxidative stress in these strains. To test this hypothesis, we measured the ROS levels upon Rv2282c, Rv1985c and Rv1990c expression in Ms . Rosup, which elevates ROS levels [ 34 ], was used as a positive control. As shown in Fig. 5 B, treatment with Rosup for 30 minutes markedly enhanced ROS levels compared with the untreated control, and overexpression of Rv2282c, Rv1985c, or Rv1990c resulted in over 50-fold increase in ROS levels relative to the empty vector control. In contrast, expression of Rv0047c, Rv3574 and Rv0602c did not significantly alter ROS levels. To further examine the relationship between tpx and oxidative stress, we overexpressed the Tpx protein in regulator-expressing strains and assessed its effect on ROS production and cell growth. Interestingly, expression of Tpx markedly reduced ROS levels (Fig. 5 C) and alleviated the growth inhibition caused by Rv2282c, Rv1985c, and Rv1990c. On the contrary, Tpx expression neither altered ROS levels (Fig. 5 C), nor restored growth in strains expressing Rv0047c, Rv3574, or Rv0602c (Fig. 5 D). Notably, none of the regulators specifically bound to the tpx promoter as assessed by EMSA assays (Supplementary Fig. 5), suggesting that the regulatory mechanism by which these regulators repress tpx expression is complicated and remains to be characterized. Together, these results show that expression of Rv2282c, Rv1985c and Rv1990c promotes the accumulation of ROS at least in part through regulating Tpx expression. Discussion In this study, we identified three transcriptional regulators, namely, Rv2282c, Rv1985c and Rv1990c, which regulate the DDR via a LexA-independent pathway in mycobacteria, and characterized that Rv2282c, Rv1985c and Rv1990c can induce oxidative stress by repressing tpx expression, and subsequently causes damage to genomic DNA, which in turn inhibits cell division and triggers activation of the DDR system in mycobacteria (Fig. 6 ). The regulators identified in this study belong to diverse transcription factor families. Rv1985c is a potential antigen for tuberculosis diagnosis and a homolog of E. coli IciA, which inhibits DNA replication [ 35 , 36 ] A recent paper showed that Rv1985c was induced in Mtb under hypoxia conditions and confirmed that Rv1985c contributes to reduced metabolic activity, thereby promoting increased tolerance to the novel tuberculosis drug bedaquiline [ 37 ]. Rv1990c, also known as MbcA, is a component of the toxin–antitoxin (TA) system that recognizes promoter regions adjacent to the LexA-binding site [ 38 ]. Its expression has been shown to be upregulated in a variety of stress conditions, including persister stress [ 39 ], hypoxic stress[ 40 ], and within host macrophages [ 41 ]. Rv2282c belongs to the LysR family of transcriptional regulators, and its function remains to be characterized. Despite their functional diversity, these regulators target the same promoter regions to control the DDR in this study, suggesting a conserved regulatory pattern in certain contexts. Transcriptional regulation of the DDR system is critical for bacterial survival. Bacteria employ multiple transcription-mediated DNA repair pathways. In addition to LexA-dependent regulatory pathway, a LexA-independent system primarily orchestrated by the transcription factor complex PafBC has been reported [ 14 , 19 , 42 ]. Previous studies have shown that these two systems recognize distinct DNA sequences. The PafBC-binding region is generally located upstream of the SOS box [ 19 ]. In this study, we found that most of these regulators control the mutasome promoter through a DNA region, approximately 80 bp upstream of the TSS (Fig. 2 C; Fig. 2 F), which shares certain similarities with the PafBC-binding pattern. A previous study suggested that PafBC binds directly to the imuAB promoter but does not show directly bind to the dnaE2 promoter [ 19 ], indicating that these regulators may act through a mechanism distinct from that of PafBC in modulating the DDR. Intracellular ROS levels critically influence the DDR system [ 43 ]. ROS can be generated through multiple pathways, including imbalances in gene expression or exposure to certain bactericidal drugs and agents [ 9 , 44 ]. Our results indicate that Rv2282c, Rv1985c and Rv1990c alter intracellular ROS levels through a Tpx-dependent manner. Tpx is a conserved mycobacterial peroxidase responsible for H₂O₂ scavenging [ 33 ]. We showed that Rv2282c, Rv1985c and Rv1990c repress tpx expression to promote ROS accumulation (Fig. 4 ; Fig. 5 ). However, Tpx is not the sole effector in this pathway, as its overexpression alone is insufficient to fully counteract the regulatory effects. Indeed, beyond tpx , we identified multiple genes involved in maintaining redox homeostasis that are also regulated by these regulators. The trx-trxB operon participates in the oxidative stress response, and Trx has been reported to enhance Tpx-mediated peroxynitrite reduction, suggesting functional interplay between these two pathways [ 11 ]. Our data show that trx-trxB is regulated by these factors, implicating this operon in the observed redox perturbation. The current investigation was conducted in Ms by overexpression method. Owing to experimental condition constraints, we did not validate these results in Mtb through gene deletion experiments. Future studies employing loss-of-function approaches in Mtb will be essential to fully elucidate the underlying regulatory mechanisms. Conclusions In summary, our work establishes a LexA-independent mode of DDR regulation in mycobacteria and links transcription factor-mediated DDR control to intracellular redox status. Our study provides a conceptual framework for understanding how mycobacteria orchestrate the complex DDR system through redox-based transcriptional regulation. Abbreviations Mtb Mycobacterium tuberculosis Ms Mycobacterium smegmatis DDR DNA damage response TR transcriptional regulator ROS reactive oxygen species ATc anhydrotetracycline EMSA electrophoretic mobility shift assay Declarations Ethics approval and consent to participate Not applicable. Clinical trial number Not applicable. Consent for publication Not applicable. Data availability The RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) with the SuperSeries accession number GSE327829（https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE327829). Competing interests The authors declare no competing interests. Funding The work was supported by the National Key Research and Development Program of China (2022YFA1303500 and 2021YFA1300901) and Major Project of Guangzhou National Laboratory (GZNL2024A01024). Authors' contributions Sige Li, Wei Zhou and Yangbo Hu designed research; Sige Li and Chun Wang performed research; Sige Li and Zhiyong Wu analyzed data; Wenjing Yu, Yong Zhang and Shiyun Chen provided materials and discussed the results; Sige Li, Xindi Huang, Shiyun Chen and Yangbo Hu wrote the manuscript; All authors commented on the manuscript. Acknowledgements We Would like to thank Dr. Zhixian Qiao and Xiaocui Chai at the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for their assistance in RNA-seq analysis. We also thank Jiahao Yan at Central China Normal University for his assistance with data analysis. References Goig GA, Windels EM, Loiseau C, Stritt C, Biru L, Borrell S, et al. Ecology, global diversity and evolutionary mechanisms in the Mycobacterium tuberculosis complex. Nat Rev Microbiol. 2025;23(9):602-14; doi: 10.1038/s41579-025-01159-w. Warner DF, Barczak AK, Gutierrez MG, Mizrahi V. Mycobacterium tuberculosis biology, pathogenicity and interaction with the host. Nat Rev Microbiol. 2025;23(12):788-804; doi: 10.1038/s41579-025-01201-x. Islam MM, Hameed HMA, Mugweru J, Chhotaray C, Wang C, Tan Y, et al. Drug resistance mechanisms and novel drug targets for tuberculosis therapy. J Genet Genomics. 2017;44(1):21-37; doi: 10.1016/j.jgg.2016.10.002. Sacchettini JC, Rubin EJ, Freundlich JS. Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. Nat Rev Microbiol. 2008;6(1):41-52; doi: 10.1038/nrmicro1816. Ahmad Khosravi N, Khosravi AD, Hashemzadeh M, Savari M, Saki M. Investigation of delamanid, bedaquiline, and linezolid resistance rates and related gene mutations in multidrug-resistant Mycobacterium tuberculosis in regional tuberculosis reference laboratories of Iran. BMC Microbiol. 2025;25(1):509; doi: 10.1186/s12866-025-04213-y. Bo H, Moure UAE, Yang Y, Pan J, Li L, Wang M, et al. Mycobacterium tuberculosis-macrophage interaction: Molecular updates. Front Cell Infect Microbiol. 2023;13:1062963; doi: 10.3389/fcimb.2023.1062963. Zhang S, Liu Y, Zhou X, Ou M, Xiao G, Li F, et al. Sirtuin 7 Regulates Nitric Oxide Production and Apoptosis to Promote Mycobacterial Clearance in Macrophages. Front Immunol. 2021;12:779235; doi: 10.3389/fimmu.2021.779235. Bulatovic VM, Wengenack NL, Uhl JR, Hall L, Roberts GD, Cockerill FR, 3rd, et al. Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid. Antimicrob Agents Chemother. 2002;46(9):2765-71; doi: 10.1128/AAC.46.9.2765-2771.2002. Dillon NA, Lamont EA, Rather MA, Baughn AD. Oxidative stress drives potent bactericidal activity of pyrazinamide against Mycobacterium tuberculosis. bioRxiv. 2024; doi: 10.1101/2024.12.17.628853. Saito K, Mishra S, Warrier T, Cicchetti N, Mi J, Weber E, et al. Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected. Sci Transl Med. 2021;13(621):eabg2612; doi: 10.1126/scitranslmed.abg2612. Trujillo M, Mauri P, Benazzi L, Comini M, De Palma A, Flohe L, et al. The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. J Biol Chem. 2006;281(29):20555-66; doi: 10.1074/jbc.M601008200. Kawai Y, Moriya S, Ogasawara N. Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis. Mol Microbiol. 2003;47(4):1113-22; doi: 10.1046/j.1365-2958.2003.03360.x. Davis EO, Dullaghan EM, Rand L. Definition of the mycobacterial SOS box and use to identify LexA-regulated genes in Mycobacterium tuberculosis. J Bacteriol. 2002;184(12):3287-95; doi: 10.1128/JB.184.12.3287-3295.2002. Muller AU, Leibundgut M, Ban N, Weber-Ban E. Structure and functional implications of WYL domain-containing bacterial DNA damage response regulator PafBC. Nat Commun. 2019;10(1):4653; doi: 10.1038/s41467-019-12567-x. Vadrevu IS, Lofton H, Sarva K, Blasczyk E, Plocinska R, Chinnaswamy J, et al. ChiZ levels modulate cell division process in mycobacteria. Tuberculosis (Edinb). 2011;91 Suppl 1(Suppl 1):S128-35; doi: 10.1016/j.tube.2011.10.022. Modell JW, Kambara TK, Perchuk BS, Laub MT. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus. PLoS Biol. 2014;12(10):e1001977; doi: 10.1371/journal.pbio.1001977. Warner DF, Ndwandwe DE, Abrahams GL, Kana BD, Machowski EE, Venclovas C, et al. Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2010;107(29):13093-8; doi: 10.1073/pnas.1002614107. Gessner S, Martin ZA, Reiche MA, Santos JA, Dinkele R, Ramudzuli A, et al. Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome. Elife. 2023;12; doi: 10.7554/eLife.75628. Adefisayo OO, Dupuy P, Nautiyal A, Bean JM, Glickman MS. Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis. Nucleic Acids Res. 2021;49(22):12805-19; doi: 10.1093/nar/gkab1169. Brzostek A, Plocinski P, Minias A, Ciszewska A, Gasior F, Pawelczyk J, et al. Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses. Cells. 2021;10(5); doi: 10.3390/cells10051168. Zhou W, Huang S, Cumming BM, Zhang Y, Tang W, Steyn AJC, et al. A Feedback Regulatory Loop Containing McdR and WhiB2 Controls Cell Division and DNA Repair in Mycobacteria. mBio. 2022;13(2):e0334321; doi: 10.1128/mbio.03343-21. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, et al. New use of BCG for recombinant vaccines. Nature. 1991;351(6326):456-60; doi: 10.1038/351456a0. Li Y, Li L, Huang L, Francis MS, Hu Y, Chen S. Yersinia Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ. Mol Microbiol. 2014;91(3):494-507; doi: 10.1111/mmi.12474. Ehrt S, Guo XV, Hickey CM, Ryou M, Monteleone M, Riley LW, et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 2005;33(2):e21; doi: 10.1093/nar/gni013. Hu Y, Wang Z, Feng L, Chen Z, Mao C, Zhu Y, et al. sigma(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria. Mol Microbiol. 2016;102(1):107-20; doi: 10.1111/mmi.13449. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884-i90; doi: 10.1093/bioinformatics/bty560. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-9; doi: 10.1038/nmeth.1923. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21; doi: 10.1093/bioinformatics/bts635. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30; doi: 10.1093/bioinformatics/btt656. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550; doi: 10.1186/s13059-014-0550-8. Cui Y, Zhao Y, Tian Y, Zhang W, Lu X, Jiang X. The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials. 2012;33(7):2327-33; doi: 10.1016/j.biomaterials.2011.11.057. Yu W, Yuan L, Zhou W, He L, Huang X, Yu J, et al. Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis. Nucleic Acids Res. 2026;54(5); doi: 10.1093/nar/gkag214. Lu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med. 2014;66:75-87; doi: 10.1016/j.freeradbiomed.2013.07.036. Ye M, Li H, Luo H, Zhou Y, Luo W, Lin Z. Potential Antioxidative Activity of Homocysteine in Erythrocytes under Oxidative Stress. Antioxidants (Basel). 2023;12(1); doi: 10.3390/antiox12010202. Chen J, Wang S, Zhang Y, Su X, Wu J, Shao L, et al. Rv1985c, a promising novel antigen for diagnosis of tuberculosis infection from BCG-vaccinated controls. BMC Infect Dis. 2010;10:273; doi: 10.1186/1471-2334-10-273. Kumar S, Farhana A, Hasnain SE. In-vitro helix opening of M. tuberculosis oriC by DnaA occurs at precise location and is inhibited by IciA like protein. PLoS One. 2009;4(1):e4139; doi: 10.1371/journal.pone.0004139. Banaei-Esfahani A, Borrell S, Trauner A, Gygli SM, Rustad TR, Feldmann J, et al. LysG-driven transcriptional network rewiring underlies lineage-specific phenotypes in Mycobacterium tuberculosis. Nat Commun. 2026; doi: 10.1038/s41467-026-70539-4. Li X, Jiang X, Xu M, Fang Y, Wang Y, Sun G, et al. Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries. AMB Express. 2020;10(1):199; doi: 10.1186/s13568-020-01133-0. Keren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. mBio. 2011;2(3):e00100-11; doi: 10.1128/mBio.00100-11. Rustad TR, Harrell MI, Liao R, Sherman DR. The enduring hypoxic response of Mycobacterium tuberculosis. PLoS One. 2008;3(1):e1502; doi: 10.1371/journal.pone.0001502. Homolka S, Niemann S, Russell DG, Rohde KH. Functional genetic diversity among Mycobacterium tuberculosis complex clinical isolates: delineation of conserved core and lineage-specific transcriptomes during intracellular survival. PLoS Pathog. 2010;6(7):e1000988; doi: 10.1371/journal.ppat.1000988. Schilling CM, Zdanowicz R, Rabl J, Muller AU, Boehringer D, Glockshuber R, et al. Single-stranded DNA binding to the transcription factor PafBC triggers the mycobacterial DNA damage response. Sci Adv. 2025;11(6):eadq9054; doi: 10.1126/sciadv.adq9054. Rath S, Das S. Oxidative stress-induced DNA damage and DNA repair mechanisms in mangrove bacteria exposed to climatic and heavy metal stressors. Environ Pollut. 2023;339:122722; doi: 10.1016/j.envpol.2023.122722. Prakash A, Dutta D. Bicyclomycin generates ROS and blocks cell division in Escherichia coli. PLoS One. 2024;19(3):e0293858; doi: 10.1371/journal.pone.0293858. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 Bacterial one hybrid analysis of genes regulating imuABp-sosmt and dnaE2p-sosmt . SupplementaryTable2.xlsx Supplementary Table 2 RNA-seq data for Ms with or without Rv2282c, Rv1985c or Rv1990c overexpression. SupplementaryTable3.xlsx Supplementary Table 3 RNA-seq data for Ms with or without Rv3574, Rv0047c or Rv0602c overexpression. SupplementaryTable4.xlsx Supplementary Table 4 The primers, strains and plasmids used in this study. Supplementaryinformation20260414.docx Supplementary Information This file contains Supplementary Figures 1-5. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviews received at journal 12 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Editor invited by journal 14 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 14 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9338042","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628163450,"identity":"d7bcfb76-da93-49bf-8d31-43b963fef420","order_by":0,"name":"Sige Li","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Sige","middleName":"","lastName":"Li","suffix":""},{"id":628163451,"identity":"75d1ed67-48f5-4981-b067-40da178860c4","order_by":1,"name":"Zhiyong Wu","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Zhiyong","middleName":"","lastName":"Wu","suffix":""},{"id":628163454,"identity":"bdb6a29a-9538-405e-8597-4d4ea91234af","order_by":2,"name":"Wei Zhou","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhou","suffix":""},{"id":628163455,"identity":"dd8bcf01-e50e-4626-89eb-1f8446659353","order_by":3,"name":"Chun Wang","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Wang","suffix":""},{"id":628163456,"identity":"6fb95972-5a7a-485f-8ecb-aa6e6c802015","order_by":4,"name":"Xindi Huang","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Xindi","middleName":"","lastName":"Huang","suffix":""},{"id":628163458,"identity":"f77d0f00-f5a5-4e08-a145-8dbc183de037","order_by":5,"name":"Wenjing Yu","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Yu","suffix":""},{"id":628163459,"identity":"c527caa8-da02-4096-8a4a-72944c6af202","order_by":6,"name":"Yong Zhang","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhang","suffix":""},{"id":628163460,"identity":"1b363c26-8956-47e0-963f-ae809a94358f","order_by":7,"name":"Shiyun Chen","email":"","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Shiyun","middleName":"","lastName":"Chen","suffix":""},{"id":628163464,"identity":"b7c2d7a5-37e5-4858-9a80-8925f9d4cb75","order_by":8,"name":"Yangbo Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYDACCR4Ghg8MBxjYSNLCOINkLcw8QC3EA4PbvcekbdvuMPCx9xgw/NxBjJY759Kkc9ueMbDxnDFg7D1DjJYbOWbSudsOM7BJpCUwM7YRq8WSdC2MYC3JB4jTInnnjLFl77/DPGw8hw8c7CVGC9/tHsMbP84clpNvb2x88JMYLQoHIDQPiDhAhAYGBvkGopSNglEwCkbBiAYAlU8z9IMkQ78AAAAASUVORK5CYII=","orcid":"","institution":"Wuhan Institute of Virology","correspondingAuthor":true,"prefix":"","firstName":"Yangbo","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2026-04-07 01:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9338042/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9338042/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107873018,"identity":"e03bee56-1f2b-4795-9da6-f65631c8bf52","added_by":"auto","created_at":"2026-04-27 08:01:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228326,"visible":true,"origin":"","legend":"\u003cp\u003eLarge-scale screening of transcriptional regulators \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein Ms\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Model of mutasome complex formation and function on damaged DNA. (\u003cstrong\u003eB\u003c/strong\u003e) Workflow of bacterial one-hybrid analysis in \u003cem\u003eE.coli\u003c/em\u003e. (\u003cstrong\u003eC\u003c/strong\u003e),\u003cstrong\u003e \u003c/strong\u003eThe screen reveals\u003cstrong\u003e \u003c/strong\u003e33 and 49 regulators that regulate \u003cem\u003eimuABp-sosmt \u003c/em\u003eand \u003cem\u003ednaE2p-sosmt\u003c/em\u003erespectively. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eExamples of bacterial one-hybrid analysis towards \u003cem\u003eimuABp-sosmt \u003c/em\u003eand \u003cem\u003ednaE2p-sosmt\u003c/em\u003e. (\u003cstrong\u003eE\u003c/strong\u003e) Workflow of \u003cem\u003emCherry\u003c/em\u003e-derived reporter analysis in \u003cem\u003eMs. \u003c/em\u003e(\u003cstrong\u003eF\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRv1985c, Rv1990c, Rv0195, Rv2282c and Rv1776c activate \u003cem\u003eimuABp-sosmt\u003c/em\u003e under ATc induction. (\u003cstrong\u003eG\u003c/strong\u003e) Rv1985c, Rv1990c, Rv0195, Rv2282c and Rv1776c activate \u003cem\u003ednaE2p-sosmt\u003c/em\u003eunder ATc induction.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/4e8a5aa34971d78d3278c1cc.png"},{"id":107872306,"identity":"88fa9aaf-2c79-4bc0-896b-ca56c395d594","added_by":"auto","created_at":"2026-04-27 07:56:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":291631,"visible":true,"origin":"","legend":"\u003cp\u003eDiverse regulation of Rv1985c, Rv1776c, Rv1990c, Rv2282c and Rv0195 on \u003cem\u003eimuAB\u003c/em\u003e promoters.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eBase mutation and\u003cstrong\u003e \u003c/strong\u003etruncated patterns of \u003cem\u003eimuAB\u003c/em\u003e promoters.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Regulation of LexA to \u003cem\u003eimuAB\u003c/em\u003ep and \u003cem\u003eimuABp-sosmt\u003c/em\u003e.(\u003cstrong\u003eC\u003c/strong\u003e) Regulation of Rv1985c, Rv1776c, Rv1990c, Rv2282c and Rv0195 to \u003cem\u003eimuABp-sosmt2 and imuABp-sosmt3\u003c/em\u003e. TR is the abbreviation of transcriptional regulator.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eBase mutation and\u003cstrong\u003e \u003c/strong\u003etruncated patterns of \u003cem\u003ednaE2\u003c/em\u003epromoters.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eE\u003c/strong\u003e) Regulation of LexA to \u003cem\u003ednaE2\u003c/em\u003ep and \u003cem\u003ednaE2p-sosmt\u003c/em\u003e. (\u003cstrong\u003eF\u003c/strong\u003e) Regulation of Rv1985c, Rv1776c, Rv1990c, Rv2282c and Rv0195 to \u003cem\u003ednaE2p-sosmt2 and dnaE2p-sosmt3\u003c/em\u003e. TR is the abbreviation of transcriptional regulator.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/ce121772b1fc7e9c13d705ca.png"},{"id":107871211,"identity":"5d60c850-ba2a-41df-be04-4e5d1ea5d3c1","added_by":"auto","created_at":"2026-04-27 07:47:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":199213,"visible":true,"origin":"","legend":"\u003cp\u003eEMSA analyses of interaction between transcriptional regulators and mutasome promoters.\u003cstrong\u003e \u003c/strong\u003eRv1985c (\u003cstrong\u003eA\u003c/strong\u003e), Rv1990c (\u003cstrong\u003eB\u003c/strong\u003e), and Rv2282c (\u003cstrong\u003eC\u003c/strong\u003e) were tested with \u003cem\u003eimuABp-sosmt2, dnaE2p-sosmt \u003c/em\u003eand\u003cem\u003e cas6\u003c/em\u003ep respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/2f62c26d11d7362a98d04eee.png"},{"id":107871241,"identity":"a6973671-3e22-4e6b-b816-628b2c717ce8","added_by":"auto","created_at":"2026-04-27 07:47:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283762,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis of the expression of mutasome-driving transcriptional regulators.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eOverview heatmap of gene variations in comparison to the vector group. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap analysis of genes associated with DNA replication and repair from cluster 2 and cluster 3. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap analysis of genes associated with cell division. (\u003cstrong\u003eD\u003c/strong\u003e) Heatmap analysis of genes associated with oxidative stress response.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eE\u003c/strong\u003e) RT-PCR quantification of differentially regulated genes from RNA-seq data. (\u003cstrong\u003eF\u003c/strong\u003e) Genes associated with cell division and oxidative stress were differentially regulated.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/c9ef7e0c4fdb4a1b58210668.png"},{"id":107871242,"identity":"ededdf4a-bd26-4abd-a812-8f18498f6684","added_by":"auto","created_at":"2026-04-27 07:47:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112551,"visible":true,"origin":"","legend":"\u003cp\u003eTpx restores the phenotype driven by transcriptional regulators.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RT-PCR quantification based on two batches of RNA-seq data. (\u003cstrong\u003eB\u003c/strong\u003e) ROS generation analysis from transcriptional regulators after ATc induction. (\u003cstrong\u003eC\u003c/strong\u003e) ROS generation analysis from transcriptional regulators with \u003cem\u003etpx\u003c/em\u003ecomplementation. (\u003cstrong\u003eD\u003c/strong\u003e) CFU counts analysis of transcriptional regulators with \u003cem\u003etpx\u003c/em\u003e complementation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/f862293d0b8f84a84ee2d4a7.png"},{"id":107872209,"identity":"642dea0e-6907-4149-931c-3758db8ea1f6","added_by":"auto","created_at":"2026-04-27 07:56:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78435,"visible":true,"origin":"","legend":"\u003cp\u003eModel for the regulation of Rv2282c, Rv1985c and Rv1990c regulation on oxidative stress, cell division and DDR. Rv2282c, Rv1985c and Rv1990c suppress \u003cem\u003etpx\u003c/em\u003e transcription, resulting in increased intracellular ROS that damages genomic DNA. This damage subsequently arrests cell division and activates the DNA damage response (DDR) machinery.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/124f061dea49a903cc1ef313.png"},{"id":108006996,"identity":"2b5e11c4-e6c5-412f-99d1-181471a2fcce","added_by":"auto","created_at":"2026-04-28 12:58:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1318565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/922c930e-fb40-4d93-9ce0-201e46ec5869.pdf"},{"id":107871220,"identity":"a2e7e24f-a7c6-40c8-b17a-a99c5a4966f3","added_by":"auto","created_at":"2026-04-27 07:47:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial one hybrid analysis of genes regulating \u003cem\u003eimuABp-sosmt\u003c/em\u003e and \u003cem\u003ednaE2p-sosmt\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/21a437bc0323b782610d4a02.xlsx"},{"id":107871335,"identity":"fc097bf9-d4cd-4a4e-857a-12d4f731b182","added_by":"auto","created_at":"2026-04-27 07:48:20","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data for \u003cem\u003eMs\u003c/em\u003e with or without Rv2282c, Rv1985c or Rv1990c overexpression.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/44c81cfd5d35ea471a94ee01.xlsx"},{"id":107872221,"identity":"fec01739-b449-4dc7-9caa-8ecafa9ab801","added_by":"auto","created_at":"2026-04-27 07:56:16","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":154315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data for \u003cem\u003eMs\u003c/em\u003e with or without Rv3574, Rv0047c or Rv0602c overexpression.\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/ab19c498ad7dd073d2bdcee2.xlsx"},{"id":107872240,"identity":"3db41963-0739-4495-893b-539dc077ba49","added_by":"auto","created_at":"2026-04-27 07:56:23","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primers, strains and plasmids used in this study.\u003c/p\u003e","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/e52db4e75850f9f7609deab4.xlsx"},{"id":107872084,"identity":"fc749a60-c83d-4c6f-ae5e-6eb9c79fe84d","added_by":"auto","created_at":"2026-04-27 07:55:23","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":30300446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis file contains Supplementary Figures 1-5.\u003c/p\u003e","description":"","filename":"Supplementaryinformation20260414.docx","url":"https://assets-eu.researchsquare.com/files/rs-9338042/v1/618963fa82a8daf29116130c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A LexA-independent redox-based transcriptional regulation to DNA Damage Response System in Mycobacteria","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (\u003cem\u003eMtb\u003c/em\u003e) represents one of the most successful pathogens, owing to its capability to persist within host macrophages and establish chronic infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With the continuous emergence of multidrug-resistant and extensively drug-resistant strains over recent decades [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the treatment of \u003cem\u003eMtb\u003c/em\u003e infection has become increasingly challenging. Host macrophages impose a variety of stresses, including oxidative stress, to eliminate infection and prevent persistent infection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Reactive oxygen species (ROS) generated by NADPH oxidase and cathepsins in macrophages, contribute to \u003cem\u003eMtb\u003c/em\u003e clearance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, certain first-line anti-tuberculosis drugs and reagents can exert bactericidal effects through their ability to induce oxidative stress in mycobacterial species and cause genomic damages [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDNA repair is intimately coupled with the regulation of cell division in bacteria. For example, in \u003cem\u003eEscherichia coli\u003c/em\u003e, SulA inhibits cell division to promote DNA repair [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In \u003cem\u003eBacillus subtilis\u003c/em\u003e, the checkpoint protein YneA prevents cell division upon DNA damage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In response to DNA damage, \u003cem\u003eMtb\u003c/em\u003e has evolved distinct DNA damage response (DDR) systems \u0026mdash;such as the SOS system and the PafBC system\u0026mdash;governed by specific transcriptional regulators to survive genotoxic stress. The SOS system is regulated by the transcriptional repressor LexA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], whereas the PafBC system is activated by the transcriptional activator complex PafBC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which directly induces gene expression upon DNA damage. In mycobacteria, the SOS and PafBC regulons both contain the checkpoint protein ChiZ, which modulates DDR and cell division to promote survival under stress conditions[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting a conserved relationship between cell division and DDR. DDR involves translesion DNA synthesis, which is performed by the mutasome complex, comprising the C-family DNA polymerase DnaE2 and its accessory factors ImuA and ImuB [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The low fidelity of the mutasome promotes survival under genotoxic stress and facilitates the emergence of drug resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As a core component of the DDR system, the mutasome is directly regulated by LexA, and is also influenced by PafBC [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous studies have indicated the existence of additional, as-yet-uncharacterized mechanisms regulating DDR-related genes, as evidenced by the observation that \u003cem\u003eMtb\u003c/em\u003e retains viability under mitomycin C stress even when both the SOS and PafBC systems are disrupted [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These findings further suggest that other transcriptional regulators are involved in DDR modulation through uncharacterized mechanisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we aimed to identify and characterize the transcriptional regulators involved in regulating the DDR systems in mycobacteria. Using a bacterial one-hybrid screening system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we revealed that Rv2282c, Rv1985c and Rv1990c activate the DDR system and inhibit cell growth through a LexA-independent pathway. These regulators drive widespread perturbation of gene expression and induce oxidative stress by regulating Tpx expression. Our data suggest that Rv2282c, Rv1985c and Rv1990c modulate cell division and DDR by regulating oxidative stress responses, revealing a previously unrecognized regulation to DDR independent of the canonical LexA regulatory pathway.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains, plasmids and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eM. smegmatis\u003c/em\u003e mc\u003csup\u003e2\u003c/sup\u003e155 (\u003cem\u003eMs\u003c/em\u003e) was cultured in Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80, 0.5% glycerol and 0.5% dextrose or on Middlebrook 7H10 agar plates supplemented with 0.5% glycerol at 37 ℃. \u003cem\u003eE. coli\u003c/em\u003e strains were cultured in Luria-Bertani (LB) broth or on LB agar plates at 37 ℃. The pMV306 were used for expressing proteins in mycobacteria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The pZT100 plasmid was used for the construction of the reporter system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and pUV15TetORm was used for the construction of overexpression plasmids [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The following antibiotics were used at the indicated concentrations: kanamycin (BiBi), 20 \u0026micro;g/mL; anhydrotetracycline (ATc; American chemistry), 50 ng/mL; streptomycin, 20 \u0026micro;g/mL on Middlebrook 7H10 agar or 10 \u0026micro;g/mL on 7H9 medium; hygromycin B, 50 \u0026micro;g/mL. Gene deletions were constructed by homologous recombination. Mutant strains were confirmed by PCR using a pair of primers located outside the cloned region, followed by sequencing of the amplified PCR products. Plasmids and primers utilized in this study were generated and listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction and RNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eFor RNA isolation, the \u003cem\u003eMs\u003c/em\u003e strains carrying the expression plasmids were grown in 7H9 broth supplemented with 50 ng/mL ATc and induced at 37 ℃ for 6 h. Cells were then collected and the total RNA was extracted from bacterial cell pellets by liquid nitrogen grinding, followed by extraction using TRIzol reagent (Invitrogen, USA) as described previously [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor RNA-seq, rRNA was depleted using the Ribo-off RNA Depletion Kit (Vazyme). RNA libraries were constructed with VAHTS Universal RNA-seq Library Prep Kit for Illumina (Vazyme). Sequencing was conducted on the Illumina HiSeq X Ten platform using 2x150bp paired-end reads. Raw reads were quality-filtered using fastp [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. rRNA reads were removed by Bowtie2 alignment against rRNA references [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Clean reads were aligned to \u003cem\u003egenome of Ms\u003c/em\u003e (NC_008596) using STAR [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and gene counts were generated with featureCounts [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Differential expression analysis was conducted using DESeq2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with significance defined as fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 and adjusted P value\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR assays\u003c/h3\u003e\n\u003cp\u003eRemoval of contaminating genomic DNA and reverse transcription were performed using the HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Vazyme, R333-01, China). The process of qRT-PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad, USA). Primers specific to target genes were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Gene-specific primers used are listed in Supplementary Table\u0026nbsp;4. Expression levels were normalized to \u003cem\u003esigA\u003c/em\u003e mRNA as the endogenous control. Data were obtained from three independent biological replicates, each measured in technical duplicate, and analyzed using CFX Manager (Bio-Rad) software.\u003c/p\u003e\n\u003ch3\u003eBacterial one-hybrid assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eimuAB\u003c/em\u003e and \u003cem\u003ednaE2\u003c/em\u003e promoters were fused to the promoter-less \u003cem\u003elacZ\u003c/em\u003e gene in pZT100 plasmid and transformed into \u003cem\u003eE. coli\u003c/em\u003e K-12 Δ\u003cem\u003elacZ\u003c/em\u003e strain to generate the reporter strains K-12 \u003cem\u003eimuAB\u003c/em\u003ep-\u003cem\u003elacZ\u003c/em\u003e and \u003cem\u003ednaE2\u003c/em\u003ep-\u003cem\u003elacZ\u003c/em\u003e respectively. The coding regions of 185 transcriptional regulators derived from \u003cem\u003eMtb\u003c/em\u003e were fused to the \u003cem\u003erpoA\u003c/em\u003e gene in pRAT103 plasmid and then transformed into the K-12 \u003cem\u003eimuAB\u003c/em\u003ep-\u003cem\u003elacZ\u003c/em\u003e and \u003cem\u003ednaE2\u003c/em\u003ep-\u003cem\u003elacZ\u003c/em\u003e strains respectively. Strains were then cultured at 30 ℃ to an OD\u003csub\u003e600\u003c/sub\u003e of ~\u0026thinsp;0.8 to test the activity of β-galactosidase as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Data were obtained from three independent biological replicates, each assayed in technical duplicate.\u003c/p\u003e\n\u003ch3\u003ePromoter activity analysis in mycobacteria\u003c/h3\u003e\n\u003cp\u003ePromoter activity in mycobacteria was analyzed as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Promoters were amplified from \u003cem\u003eMtb\u003c/em\u003e H37Rv genomic DNA using the specific primers (Supplementary Table\u0026nbsp;4) and were fused to a promoter-less \u003cem\u003emCherry\u003c/em\u003e gene on the pMV306 plasmid. Recombinant plasmids were co-electroporated with the transcriptional regulator-expressing plasmid pUV15TetORm into \u003cem\u003eMs\u003c/em\u003e wild-type strain. Cells were cultured at 37 ℃ in 7H9 broth to an OD\u003csub\u003e600\u003c/sub\u003e of ~\u0026thinsp;0.3, followed by the incubation with 50 ng/mL ATc. Cells were sampled, and promoter activity was quantified as relative fluorescence units (RFU; fluorescence intensities per OD₆₀₀ unit) using a BioTek Synergy H1 microplate reader. All assays were performed in three independent experiments.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGrowth assay of the strains expressing the transcriptional regulator\u003c/h2\u003e \u003cp\u003eTranscriptional regulators were amplified from \u003cem\u003eMtb\u003c/em\u003e H37Rv genomic DNA using the specific primers (Supplementary Table\u0026nbsp;4) and cloned into pUV15TetORm. Recombinant plasmids were electroporated into \u003cem\u003eMs\u003c/em\u003e strain. Cells were cultured to mid-logarithmic phase in 7H9 broth at 37 ℃, followed by the incubation with 50 ng/mL ATc. Cells were sampled every 3 h, and optical density at 600 nm (OD₆₀₀) was measured using a BioTek Synergy H1 microplate reader. All assays were performed in three independent experiments.\u003c/p\u003e \u003cp\u003eFor growth on agar plates, cells were grown to an OD\u003csub\u003e600\u003c/sub\u003e of ~\u0026thinsp;0.4, serially 10-fold diluted, and spotted onto agar plates supplemented with hygromycin B and ATc. Plates without ATc were served as a negative control. All plates were cultured at 37 ℃ for 3 days and imaged using an Epson imaging system. Assays were performed in two independent experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of reactive oxygen species\u003c/h3\u003e\n\u003cp\u003eIntracellular ROS levels were quantified using a Reactive Oxygen Species Assay Kit (Beyotime, China) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eMs\u003c/em\u003e strains carrying pUV15TetORm plasmid were cultured at OD600 of ~\u0026thinsp;0.3, and ATc was then added to a final concentration of 50 ng/mL to induce for approximately 6 h. Rosup (Beyotime, China) was added at the dilution of 1:1000 as positive control to induce the ROS. Cells were then collected and pellets were washed and centrifuged three times in 7H9 broth with 50 \u0026micro;g/mL hygromycin. Cells were then resuspended in 7H9 medium with 50 \u0026micro;g/mL hygromycin, and DCFH-DA was added to each sample at a 1:1000 dilution. Cultures were then incubated at 37 ℃ away from light for approximately 0.5-1 h. 7H9 broth containing 50 \u0026micro;g/mL hygromycin was then used to wash cells at least three times to remove residual dye. Cultures were finally resuspended and fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Serial dilutions of cells were plated on agar plates and incubated at 37 ℃ for 3 d. Colonies were counted to determine viable cell counts. Assays were performed in three independent experiments.\u003c/p\u003e\n\u003ch3\u003eProtein expression and purification\u003c/h3\u003e\n\u003cp\u003eTranscriptional regulator genes were amplified from \u003cem\u003eMtb\u003c/em\u003e H37Rv genomic DNA using the specific primers (Supplementary Table\u0026nbsp;4) and cloned into pET21a. For protein expression, recombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), and strains were grown at 37 ℃ to an OD\u003csub\u003e600\u003c/sub\u003e of ~\u0026thinsp;0.6. Expression was induced with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 ℃ for 20 h [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Cell pellets were collected and resuspended lysis buffer (40 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.9). Suspensions were disrupted by sonication and clarified by high-speed centrifugation. The supernatant was collected and mixed with a prepacked His column, and non-specifically bound proteins were removed by washing with wash buffer (40 mM Tris, 300 mM NaCl, 10% glycine and 50 mM imidazole (pH 7.9). Target proteins were eluted with elution buffer (40 mM Tris, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 7.9). Eluted fractions were collected and analyzed by SDS-PAGE.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA-binding analysis\u003c/h2\u003e \u003cp\u003eEMSA was performed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, 200\u0026ndash;300 bp FAM-labeled promoter fragments were amplified by PCR and then purified using a gel extraction kit (Omega). Promoter fragments (30 nM) were incubated with purified transcriptional regulator proteins in TB buffer (20 mM Tris - HCl, pH 7.9, 50 mM NaCl, 5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1 mM dithiothreitol, 0.1 mM EDTA, 5% glycerol) at 37\u0026deg;C for 15\u0026ndash;30 min. Samples were resolved on a 6% native 0.5 \u0026times; TBE-PAGE gel at 100 V for 1 h. Gels were scanned using an Amersham Typhoon imager (GE Healthcare).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe p values shown were calculated using two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test: no significance (ns), p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; \u003csup\u003e*\u003c/sup\u003e, p \u003cem\u003e⩽\u003c/em\u003e 0.05; \u003csup\u003e**\u003c/sup\u003e, p \u003cem\u003e⩽\u003c/em\u003e 0.01; \u003csup\u003e***\u003c/sup\u003e, p \u003cem\u003e⩽\u003c/em\u003e 0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eScreening transcriptional regulators of DDR system\u003c/h2\u003e \u003cp\u003eTo identify transcriptional regulators that regulate the DDR system, we performed an \u003cem\u003ein vitro\u003c/em\u003e large-scale bacterial one-hybrid screen using a library of 185 transcriptional regulators from \u003cem\u003eMtb\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As ImuAB and DnaE2 function as the mutasome complex in the DDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we used both \u003cem\u003eimuAB\u003c/em\u003e and \u003cem\u003ednaE2\u003c/em\u003e promoters derived from \u003cem\u003eMtb\u003c/em\u003e H37Rv as targets for regulator screening. Their SOS-box sequences were mutated to abolish LexA-mediated regulation and were named as \u003cem\u003eimuABp-sosmt\u003c/em\u003e and \u003cem\u003ednaE2p-sosmt\u003c/em\u003e respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Each promoter was transcriptionally fused to a \u003cem\u003elacZ\u003c/em\u003e reporter gene, and the regulatory activity was quantified by measuring the β-galactosidase activity in \u003cem\u003eE. coli\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The Bacterial one-hybrid screening revealed 69 transcription factors capable of regulating the SOS-box-mutated mutasome promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Table\u0026nbsp;1). Certain regulators are indicated to be involved in regulating \u003cem\u003eimuABp\u003c/em\u003e-\u003cem\u003esosmt\u003c/em\u003e and \u003cem\u003ednaE2p\u003c/em\u003e-\u003cem\u003esosmt\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs ImuAB functions together with DnaE2 in the DDR, we next examined whether these regulators would regulate both promoters using a mycobacterial fluorescent reporter system. Briefly, \u003cem\u003eimuABp-sosmt\u003c/em\u003e or \u003cem\u003ednaE2p-sosmt\u003c/em\u003e was fused to a \u003cem\u003emCherry\u003c/em\u003e fluorescence gene, and each of the four candidate regulators was expressed in \u003cem\u003eMs\u003c/em\u003e under anhydrotetracycline (ATc) induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Interestingly, overexpression of Rv2282c, Rv0195, Rv1985c, Rv1776c, or Rv1990c in \u003cem\u003eMs\u003c/em\u003e activated both \u003cem\u003eimuABp-sosmt\u003c/em\u003e and \u003cem\u003ednaE2p-sosmt\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003eFurthermore, we observed that overexpression of these regulators uniformly inhibited cell growth both in liquid medium and on agar plates (Supplementary Fig.\u0026nbsp;1A, B). These data suggest that Rv2282c, Rv0195, Rv1985c, Rv1776c and Rv1990c can activate the mutasome complex from DDR system and are associated with growth inhibition in mycobacteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRv2282c, Rv0195, Rv1985c, Rv1776c and Rv1990c regulate mutasome in a LexA-independent manner\u003c/h2\u003e \u003cp\u003eTo characterize the regulatory mechanisms of the identified transcriptional activators, we performed a promoter mutation and truncation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For \u003cem\u003ethe imuAB\u003c/em\u003e promoter, we first tested whether the \u003cem\u003eimuABp-sosmt\u003c/em\u003e promoter was LexA-independent. Compared with the wild-type promoter, mutation of the SOS-box motif increased the promoter activity by over 10-fold and abolished LexA-mediated repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), confirming the SOS-box mutation in \u003cem\u003eimuABp-sosmt\u003c/em\u003e promoter effectively eliminated LexA regulation. We then truncated the \u003cem\u003eimuABp-sosmt\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and found that Rv2282c Rv0195 and Rv1985c still activated \u003cem\u003eimuABp-sosmt2\u003c/em\u003e but had no effect on \u003cem\u003eimuABp-sosmt3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that the region from \u0026minus;\u0026thinsp;138 to -88 upstream of the transcriptional start site (TSS) is critical for their regulation. In contrast, Rv1776c did not activate \u003cem\u003eimuABp-sosmt2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), implying its regulatory effect is distinct from other regulators. Rv1990c retained regulatory activity toward \u003cem\u003eimuABp-sosmt3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that its recognition site lies proximal to the SOS-box.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the \u003cem\u003ednaE2\u003c/em\u003e promoter, we also performed promoter truncation and mutation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, LexA similarly failed to repress the SOS-box-mutated \u003cem\u003ednaE2p-sosmt\u003c/em\u003e promoter. Rv2282c, Rv0195, Rv1990c and Rv1776c lost their regulatory activity when the promoter was truncated to \u003cem\u003ednaE2p-sosmt2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), thereby their recognition sites are localized in the region from \u0026minus;\u0026thinsp;192 to -136 upstream of the TSS. Rv1985c activated \u003cem\u003ednaE2p-sosmt2\u003c/em\u003e, its recognition site lies in the region\u0026thinsp;\u0026minus;\u0026thinsp;136 to -82 upstream of the TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eTogether, we conclude that, although these regulators activate the mutasome promoters through different DNA regions, they all act in a LexA-independent pathway, as their regulatory activity requires a DNA region upstream of promoter, and mutation of the SOS-box did not influence the regulatory effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNon-specific binding to mutasome promoters by Rv2282c, Rv1990c and Rv1985c\u003c/h2\u003e \u003cp\u003eTo investigate whether these regulators directly bind to the mutasome promoters, we performed electrophoretic mobility shift assay (EMSA). Rv2282c, Rv1990c and Rv1985c were successfully purified (Supplementary Fig.\u0026nbsp;2), whereas Rv0195 could not be obtained despite multiple attempts under various purification conditions. As these three regulators regulated both \u003cem\u003eimuABp-sosmt2\u003c/em\u003e and \u003cem\u003ednaE2p-sosmt\u003c/em\u003e, we select these two promoters for EMSA analysis. An unrelated promoter \u003cem\u003ecas6\u003c/em\u003ep [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] was selected as a negative control. Rv1985c and Rv1990c displayed binding affinity to the mutasome promoters and to \u003cem\u003ecas6\u003c/em\u003ep with comparable affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), indicating that these interactions are likely nonspecific. Rv2282c showed no detectable binding to either mutasome promoter or \u003cem\u003ecas6\u003c/em\u003ep (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting that Rv2282c may not directly bind mutasome promoter DNA. In conclusion, these data indicate that Rv2282c, Rv1990c and Rv1985c may not specifically bind to mutasome promoters and may regulate DDR system through indirect way.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRegulatory targets of Rv2282c, Rv1985c and Rv1990c\u003c/h2\u003e \u003cp\u003eTo identify the downstream targets of these transcriptional regulators, we conducted the RNA-seq analysis in \u003cem\u003eMs\u003c/em\u003e strains overexpressing either Rv2282c, Rv1985c or Rv1990c. Induction of each regulator altered the expression of ~\u0026thinsp;25% of all genes compared with the strain carrying the empty vector control (Supplementary Table\u0026nbsp;2). Differentially expressed genes were grouped into five clusters based on their expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Genes associated with DNA replication and damage response were mainly distributed in cluster 3, and were markedly activated by all of these three regulators (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;3). In contrast, genes involved in cell division were mainly assigned to cluster 5 and were greatly repressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Oxidative stress-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), such as the \u003cem\u003etrx-trxB\u003c/em\u003e operon, which encodes a thioredoxin system involved in redox balance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], were upregulated, indicating that Rv2282c, Rv1985c and Rv1990c may perturb the redox homeostasis of mycobacteria. We next performed quantitative real-time PCR (qRT-PCR) assays to validate the RNA-seq results. Each transcript was normalized to the expression level of an essential RNA polymerase sigma factor, \u003cem\u003esigA\u003c/em\u003e. Consistent with the RNA-seq data, genes associated with DDR and oxidative stress showed enhanced expression compared to the empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs these regulators all inhibited mycobacterial growth, we next examined whether DDR regulation was mediated by growth arrest. We therefore selected Rv0047c, Rv3574 and Rv0602c, which also inhibited the growth of \u003cem\u003eMs\u003c/em\u003e for further RNA-seq analysis (Supplementary Fig.\u0026nbsp;4). Genes involved cell division were also repressed by these regulators, but genes involved in oxidative stress were differentially regulated by the DDR-activating regulators (Rv2282c, Rv1985c and Rv1990c), but not by Rv0047c, Rv3574 or Rv0602c (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; Supplementary Table\u0026nbsp;3). Together, our data suggest that Rv2282c, Rv1985c and Rv1990c activate of the DDR by reprogramming of redox-associated gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTpx relieves oxidative stress and growth inhibition induced by Rv2282c, Rv1985c and Rv1990c\u003c/h2\u003e \u003cp\u003eRNA-seq analysis revealed that oxidative stress response may be involved in DDR regulation. Notably, the thiol peroxidase Tpx (encoded by \u003cem\u003eMSMEG_3479\u003c/em\u003e), which is involved in ROS detoxification, was downregulated by Rv2282c, Rv1985c and Rv1990c but not by the control regulators Rv0047c, Rv3574 and Rv0602c (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A previous study showed that Tpx is an efficient peroxidase to eliminate ROS in bacteria [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], indicating that reduced Tpx expression may be associated with the increased oxidative stress in these strains. To test this hypothesis, we measured the ROS levels upon Rv2282c, Rv1985c and Rv1990c expression in \u003cem\u003eMs\u003c/em\u003e. Rosup, which elevates ROS levels [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], was used as a positive control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, treatment with Rosup for 30 minutes markedly enhanced ROS levels compared with the untreated control, and overexpression of Rv2282c, Rv1985c, or Rv1990c resulted in over 50-fold increase in ROS levels relative to the empty vector control. In contrast, expression of Rv0047c, Rv3574 and Rv0602c did not significantly alter ROS levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine the relationship between \u003cem\u003etpx\u003c/em\u003e and oxidative stress, we overexpressed the Tpx protein in regulator-expressing strains and assessed its effect on ROS production and cell growth. Interestingly, expression of Tpx markedly reduced ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and alleviated the growth inhibition caused by Rv2282c, Rv1985c, and Rv1990c. On the contrary, Tpx expression neither altered ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), nor restored growth in strains expressing Rv0047c, Rv3574, or Rv0602c (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, none of the regulators specifically bound to the \u003cem\u003etpx\u003c/em\u003e promoter as assessed by EMSA assays (Supplementary Fig.\u0026nbsp;5), suggesting that the regulatory mechanism by which these regulators repress \u003cem\u003etpx\u003c/em\u003e expression is complicated and remains to be characterized. Together, these results show that expression of Rv2282c, Rv1985c and Rv1990c promotes the accumulation of ROS at least in part through regulating Tpx expression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified three transcriptional regulators, namely, Rv2282c, Rv1985c and Rv1990c, which regulate the DDR via a LexA-independent pathway in mycobacteria, and characterized that Rv2282c, Rv1985c and Rv1990c can induce oxidative stress by repressing \u003cem\u003etpx\u003c/em\u003e expression, and subsequently causes damage to genomic DNA, which in turn inhibits cell division and triggers activation of the DDR system in mycobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe regulators identified in this study belong to diverse transcription factor families. Rv1985c is a potential antigen for tuberculosis diagnosis and a homolog of \u003cem\u003eE. coli\u003c/em\u003e IciA, which inhibits DNA replication [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] A recent paper showed that Rv1985c was induced in \u003cem\u003eMtb\u003c/em\u003e under hypoxia conditions and confirmed that Rv1985c contributes to reduced metabolic activity, thereby promoting increased tolerance to the novel tuberculosis drug bedaquiline [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Rv1990c, also known as MbcA, is a component of the toxin\u0026ndash;antitoxin (TA) system that recognizes promoter regions adjacent to the LexA-binding site [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Its expression has been shown to be upregulated in a variety of stress conditions, including persister stress [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], hypoxic stress[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and within host macrophages [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Rv2282c belongs to the LysR family of transcriptional regulators, and its function remains to be characterized. Despite their functional diversity, these regulators target the same promoter regions to control the DDR in this study, suggesting a conserved regulatory pattern in certain contexts.\u003c/p\u003e \u003cp\u003eTranscriptional regulation of the DDR system is critical for bacterial survival. Bacteria employ multiple transcription-mediated DNA repair pathways. In addition to LexA-dependent regulatory pathway, a LexA-independent system primarily orchestrated by the transcription factor complex PafBC has been reported [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Previous studies have shown that these two systems recognize distinct DNA sequences. The PafBC-binding region is generally located upstream of the SOS box [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, we found that most of these regulators control the mutasome promoter through a DNA region, approximately 80 bp upstream of the TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), which shares certain similarities with the PafBC-binding pattern. A previous study suggested that PafBC binds directly to the \u003cem\u003eimuAB\u003c/em\u003e promoter but does not show directly bind to the \u003cem\u003ednaE2\u003c/em\u003e promoter [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], indicating that these regulators may act through a mechanism distinct from that of PafBC in modulating the DDR.\u003c/p\u003e \u003cp\u003eIntracellular ROS levels critically influence the DDR system [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. ROS can be generated through multiple pathways, including imbalances in gene expression or exposure to certain bactericidal drugs and agents [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our results indicate that Rv2282c, Rv1985c and Rv1990c alter intracellular ROS levels through a Tpx-dependent manner. Tpx is a conserved mycobacterial peroxidase responsible for H₂O₂ scavenging [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We showed that Rv2282c, Rv1985c and Rv1990c repress \u003cem\u003etpx\u003c/em\u003e expression to promote ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, Tpx is not the sole effector in this pathway, as its overexpression alone is insufficient to fully counteract the regulatory effects. Indeed, beyond \u003cem\u003etpx\u003c/em\u003e, we identified multiple genes involved in maintaining redox homeostasis that are also regulated by these regulators. The \u003cem\u003etrx-trxB\u003c/em\u003e operon participates in the oxidative stress response, and Trx has been reported to enhance Tpx-mediated peroxynitrite reduction, suggesting functional interplay between these two pathways [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our data show that \u003cem\u003etrx-trxB\u003c/em\u003e is regulated by these factors, implicating this operon in the observed redox perturbation.\u003c/p\u003e \u003cp\u003eThe current investigation was conducted in \u003cem\u003eMs\u003c/em\u003e by overexpression method. Owing to experimental condition constraints, we did not validate these results in \u003cem\u003eMtb\u003c/em\u003e through gene deletion experiments. Future studies employing loss-of-function approaches in \u003cem\u003eMtb\u003c/em\u003e will be essential to fully elucidate the underlying regulatory mechanisms.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our work establishes a LexA-independent mode of DDR regulation in mycobacteria and links transcription factor-mediated DDR control to intracellular redox status. Our study provides a conceptual framework for understanding how mycobacteria orchestrate the complex DDR system through redox-based transcriptional regulation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMtb\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDDR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDNA damage response\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranscriptional regulator\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eATc\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanhydrotetracycline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEMSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectrophoretic mobility shift assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eClinical trial number\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) with the SuperSeries accession number GSE327829（https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE327829).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Key Research and Development Program of China (2022YFA1303500 and 2021YFA1300901) and Major Project of Guangzhou National Laboratory (GZNL2024A01024).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eSige Li, Wei Zhou and Yangbo Hu designed research; Sige Li and Chun Wang performed research; Sige Li and Zhiyong Wu analyzed data; Wenjing Yu, Yong Zhang and Shiyun Chen provided materials and discussed the results; Sige Li, Xindi Huang, Shiyun Chen and Yangbo Hu wrote the manuscript; All authors commented on the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe Would like to thank Dr. Zhixian Qiao and Xiaocui Chai at the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for their assistance in RNA-seq analysis. We also thank Jiahao Yan at Central China Normal University for his assistance with data analysis.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGoig GA, Windels EM, Loiseau C, Stritt C, Biru L, Borrell S, et al. Ecology, global diversity and evolutionary mechanisms in the Mycobacterium tuberculosis complex. Nat Rev Microbiol. 2025;23(9):602-14; doi: 10.1038/s41579-025-01159-w.\u003c/li\u003e\n \u003cli\u003eWarner DF, Barczak AK, Gutierrez MG, Mizrahi V. Mycobacterium tuberculosis biology, pathogenicity and interaction with the host. Nat Rev Microbiol. 2025;23(12):788-804; doi: 10.1038/s41579-025-01201-x.\u003c/li\u003e\n \u003cli\u003eIslam MM, Hameed HMA, Mugweru J, Chhotaray C, Wang C, Tan Y, et al. Drug resistance mechanisms and novel drug targets for tuberculosis therapy. J Genet Genomics. 2017;44(1):21-37; doi: 10.1016/j.jgg.2016.10.002.\u003c/li\u003e\n \u003cli\u003eSacchettini JC, Rubin EJ, Freundlich JS. Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. Nat Rev Microbiol. 2008;6(1):41-52; doi: 10.1038/nrmicro1816.\u003c/li\u003e\n \u003cli\u003eAhmad Khosravi N, Khosravi AD, Hashemzadeh M, Savari M, Saki M. Investigation of delamanid, bedaquiline, and linezolid resistance rates and related gene mutations in multidrug-resistant Mycobacterium tuberculosis in regional tuberculosis reference laboratories of Iran. BMC Microbiol. 2025;25(1):509; doi: 10.1186/s12866-025-04213-y.\u003c/li\u003e\n \u003cli\u003eBo H, Moure UAE, Yang Y, Pan J, Li L, Wang M, et al. Mycobacterium tuberculosis-macrophage interaction: Molecular updates. Front Cell Infect Microbiol. 2023;13:1062963; doi: 10.3389/fcimb.2023.1062963.\u003c/li\u003e\n \u003cli\u003eZhang S, Liu Y, Zhou X, Ou M, Xiao G, Li F, et al. Sirtuin 7 Regulates Nitric Oxide Production and Apoptosis to Promote Mycobacterial Clearance in Macrophages. Front Immunol. 2021;12:779235; doi: 10.3389/fimmu.2021.779235.\u003c/li\u003e\n \u003cli\u003eBulatovic VM, Wengenack NL, Uhl JR, Hall L, Roberts GD, Cockerill FR, 3rd, et al. Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid. Antimicrob Agents Chemother. 2002;46(9):2765-71; doi: 10.1128/AAC.46.9.2765-2771.2002.\u003c/li\u003e\n \u003cli\u003eDillon NA, Lamont EA, Rather MA, Baughn AD. Oxidative stress drives potent bactericidal activity of pyrazinamide against Mycobacterium tuberculosis. bioRxiv. 2024; doi: 10.1101/2024.12.17.628853.\u003c/li\u003e\n \u003cli\u003eSaito K, Mishra S, Warrier T, Cicchetti N, Mi J, Weber E, et al. Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected. Sci Transl Med. 2021;13(621):eabg2612; doi: 10.1126/scitranslmed.abg2612.\u003c/li\u003e\n \u003cli\u003eTrujillo M, Mauri P, Benazzi L, Comini M, De Palma A, Flohe L, et al. The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. J Biol Chem. 2006;281(29):20555-66; doi: 10.1074/jbc.M601008200.\u003c/li\u003e\n \u003cli\u003eKawai Y, Moriya S, Ogasawara N. Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis. Mol Microbiol. 2003;47(4):1113-22; doi: 10.1046/j.1365-2958.2003.03360.x.\u003c/li\u003e\n \u003cli\u003eDavis EO, Dullaghan EM, Rand L. Definition of the mycobacterial SOS box and use to identify LexA-regulated genes in Mycobacterium tuberculosis. J Bacteriol. 2002;184(12):3287-95; doi: 10.1128/JB.184.12.3287-3295.2002.\u003c/li\u003e\n \u003cli\u003eMuller AU, Leibundgut M, Ban N, Weber-Ban E. Structure and functional implications of WYL domain-containing bacterial DNA damage response regulator PafBC. Nat Commun. 2019;10(1):4653; doi: 10.1038/s41467-019-12567-x.\u003c/li\u003e\n \u003cli\u003eVadrevu IS, Lofton H, Sarva K, Blasczyk E, Plocinska R, Chinnaswamy J, et al. ChiZ levels modulate cell division process in mycobacteria. Tuberculosis (Edinb). 2011;91 Suppl 1(Suppl 1):S128-35; doi: 10.1016/j.tube.2011.10.022.\u003c/li\u003e\n \u003cli\u003eModell JW, Kambara TK, Perchuk BS, Laub MT. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus. PLoS Biol. 2014;12(10):e1001977; doi: 10.1371/journal.pbio.1001977.\u003c/li\u003e\n \u003cli\u003eWarner DF, Ndwandwe DE, Abrahams GL, Kana BD, Machowski EE, Venclovas C, et al. Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2010;107(29):13093-8; doi: 10.1073/pnas.1002614107.\u003c/li\u003e\n \u003cli\u003eGessner S, Martin ZA, Reiche MA, Santos JA, Dinkele R, Ramudzuli A, et al. Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome. Elife. 2023;12; doi: 10.7554/eLife.75628.\u003c/li\u003e\n \u003cli\u003eAdefisayo OO, Dupuy P, Nautiyal A, Bean JM, Glickman MS. Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis. Nucleic Acids Res. 2021;49(22):12805-19; doi: 10.1093/nar/gkab1169.\u003c/li\u003e\n \u003cli\u003eBrzostek A, Plocinski P, Minias A, Ciszewska A, Gasior F, Pawelczyk J, et al. Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses. Cells. 2021;10(5); doi: 10.3390/cells10051168.\u003c/li\u003e\n \u003cli\u003eZhou W, Huang S, Cumming BM, Zhang Y, Tang W, Steyn AJC, et al. A Feedback Regulatory Loop Containing McdR and WhiB2 Controls Cell Division and DNA Repair in Mycobacteria. mBio. 2022;13(2):e0334321; doi: 10.1128/mbio.03343-21.\u003c/li\u003e\n \u003cli\u003eStover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, et al. New use of BCG for recombinant vaccines. Nature. 1991;351(6326):456-60; doi: 10.1038/351456a0.\u003c/li\u003e\n \u003cli\u003eLi Y, Li L, Huang L, Francis MS, Hu Y, Chen S. Yersinia Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ. Mol Microbiol. 2014;91(3):494-507; doi: 10.1111/mmi.12474.\u003c/li\u003e\n \u003cli\u003eEhrt S, Guo XV, Hickey CM, Ryou M, Monteleone M, Riley LW, et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 2005;33(2):e21; doi: 10.1093/nar/gni013.\u003c/li\u003e\n \u003cli\u003eHu Y, Wang Z, Feng L, Chen Z, Mao C, Zhu Y, et al. sigma(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria. Mol Microbiol. 2016;102(1):107-20; doi: 10.1111/mmi.13449.\u003c/li\u003e\n \u003cli\u003eChen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884-i90; doi: 10.1093/bioinformatics/bty560.\u003c/li\u003e\n \u003cli\u003eLangmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-9; doi: 10.1038/nmeth.1923.\u003c/li\u003e\n \u003cli\u003eDobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21; doi: 10.1093/bioinformatics/bts635.\u003c/li\u003e\n \u003cli\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30; doi: 10.1093/bioinformatics/btt656.\u003c/li\u003e\n \u003cli\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550; doi: 10.1186/s13059-014-0550-8.\u003c/li\u003e\n \u003cli\u003eCui Y, Zhao Y, Tian Y, Zhang W, Lu X, Jiang X. The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials. 2012;33(7):2327-33; doi: 10.1016/j.biomaterials.2011.11.057.\u003c/li\u003e\n \u003cli\u003eYu W, Yuan L, Zhou W, He L, Huang X, Yu J, et al. Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis. Nucleic Acids Res. 2026;54(5); doi: 10.1093/nar/gkag214.\u003c/li\u003e\n \u003cli\u003eLu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med. 2014;66:75-87; doi: 10.1016/j.freeradbiomed.2013.07.036.\u003c/li\u003e\n \u003cli\u003eYe M, Li H, Luo H, Zhou Y, Luo W, Lin Z. Potential Antioxidative Activity of Homocysteine in Erythrocytes under Oxidative Stress. Antioxidants (Basel). 2023;12(1); doi: 10.3390/antiox12010202.\u003c/li\u003e\n \u003cli\u003eChen J, Wang S, Zhang Y, Su X, Wu J, Shao L, et al. Rv1985c, a promising novel antigen for diagnosis of tuberculosis infection from BCG-vaccinated controls. BMC Infect Dis. 2010;10:273; doi: 10.1186/1471-2334-10-273.\u003c/li\u003e\n \u003cli\u003eKumar S, Farhana A, Hasnain SE. In-vitro helix opening of M. tuberculosis oriC by DnaA occurs at precise location and is inhibited by IciA like protein. PLoS One. 2009;4(1):e4139; doi: 10.1371/journal.pone.0004139.\u003c/li\u003e\n \u003cli\u003eBanaei-Esfahani A, Borrell S, Trauner A, Gygli SM, Rustad TR, Feldmann J, et al. LysG-driven transcriptional network rewiring underlies lineage-specific phenotypes in Mycobacterium tuberculosis. Nat Commun. 2026; doi: 10.1038/s41467-026-70539-4.\u003c/li\u003e\n \u003cli\u003eLi X, Jiang X, Xu M, Fang Y, Wang Y, Sun G, et al. Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries. AMB Express. 2020;10(1):199; doi: 10.1186/s13568-020-01133-0.\u003c/li\u003e\n \u003cli\u003eKeren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. mBio. 2011;2(3):e00100-11; doi: 10.1128/mBio.00100-11.\u003c/li\u003e\n \u003cli\u003eRustad TR, Harrell MI, Liao R, Sherman DR. The enduring hypoxic response of Mycobacterium tuberculosis. PLoS One. 2008;3(1):e1502; doi: 10.1371/journal.pone.0001502.\u003c/li\u003e\n \u003cli\u003eHomolka S, Niemann S, Russell DG, Rohde KH. Functional genetic diversity among Mycobacterium tuberculosis complex clinical isolates: delineation of conserved core and lineage-specific transcriptomes during intracellular survival. PLoS Pathog. 2010;6(7):e1000988; doi: 10.1371/journal.ppat.1000988.\u003c/li\u003e\n \u003cli\u003eSchilling CM, Zdanowicz R, Rabl J, Muller AU, Boehringer D, Glockshuber R, et al. Single-stranded DNA binding to the transcription factor PafBC triggers the mycobacterial DNA damage response. Sci Adv. 2025;11(6):eadq9054; doi: 10.1126/sciadv.adq9054.\u003c/li\u003e\n \u003cli\u003eRath S, Das S. Oxidative stress-induced DNA damage and DNA repair mechanisms in mangrove bacteria exposed to climatic and heavy metal stressors. Environ Pollut. 2023;339:122722; doi: 10.1016/j.envpol.2023.122722.\u003c/li\u003e\n \u003cli\u003ePrakash A, Dutta D. Bicyclomycin generates ROS and blocks cell division in Escherichia coli. PLoS One. 2024;19(3):e0293858; doi: 10.1371/journal.pone.0293858.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mycobacteria, DNA damage response, Mutasome, ROS, Transcriptional regulation","lastPublishedDoi":"10.21203/rs.3.rs-9338042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9338042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eThe bacterial DNA damage response (DDR) system promotes survival under stressful conditions and facilitates the emergence of drug-resistant mutations. The DDR system in \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e is primarily governed by LexA-mediated transcriptional repression, yet whether additional regulatory inputs contribute to DDR remains to be characterized.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eBy performing a bacterial one-hybrid screen, we identified several transcriptional regulators, including Rv2282c, Rv1985c and Rv1990c, that activate DDR gene expression and inhibit cell growth. Promoter truncation analysis revealed that these regulators act through sequences distinct from the canonical LexA-binding motif. No specific interaction between these regulators and DDR gene promoters was \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edetected by\u003c/span\u003e EMSAs, indicating an indirect regulatory mechanism. RNA-seq analysis revealed that approximately 25% genes were differentially expressed upon overexpression of each of these regulators. Among these, \u003cem\u003etpx\u003c/em\u003e, which is responsible for oxidative stress defense, was consistently repressed by all of the three DDR-activating regulators but not by control regulators. Complementation of Tpx alleviated ROS accumulation and restored cell growth in strains overexpressing these regulators, suggesting that the DDR activation is linked to redox imbalance caused by Tpx repression.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eOur data demonstrate that Rv2282c, Rv1985c, and Rv1990c activate the DDR through perturbation of redox homeostasis, revealing a previously unrecognized, LexA-independent regulation pathway in mycobacteria.\u003c/p\u003e","manuscriptTitle":"A LexA-independent redox-based transcriptional regulation to DNA Damage Response System in Mycobacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 06:03:37","doi":"10.21203/rs.3.rs-9338042/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T02:54:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T20:35:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T13:21:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193686610099257765072902723774616240553","date":"2026-04-23T06:31:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64539552011925610734832956542040864763","date":"2026-04-23T02:35:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68619294017040338229475759983798156336","date":"2026-04-21T14:07:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T19:06:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T03:46:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-15T03:33:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T08:10:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-04-14T07:50:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e258b54a-9b5a-42c1-a7c0-381fb7c6e4cd","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T02:54:49+00:00","index":35,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T20:35:39+00:00","index":34,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T13:21:38+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T06:03:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 06:03:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9338042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9338042","identity":"rs-9338042","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}