InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG

Isoniazid (INH), an anti-tuberculosis (TB) drug, has been widely used for nearly 60 years. However, the pathway through which Mycobacterium tuberculosis responds INH remain largely unclear. In this study, we characterized a novel transcriptional factor, InbR, which is encoded by Rv0275c and belongs to the TetR family, that is directly responsive to INH. Disrupting inbR made mycobacteria more sensitive to INH, whereas overexpressing inbR decreased bacterial susceptibility to the drug. InbR could bind specifically to the upstream region of its own operon at two inverted repeats and act as an auto-repressor. Furthermore, InbR directly bind with INH, and the binding reduced InbR’s DNA-binding ability. Interestingly, susceptibilities were also changed by InbR for other anti-TB drugs, such as rifampin, implying that InbR may play a role in multi-drug resistance. Additionally, microarray analyses revealed a portion genes of the inbR regulon have similar expression patterns in inbR-overexpressing strain and INH-treated wild type strain, suggesting that these genes, for example iniBAC, may be responsible to the drug resistance of inbR-overexpressing strain. The regulation of these genes by InbR were further assessed by ChIP-seq assay. InbR may regulate multiple drug resistance of mycobacteria through the regulation of these genes.

Scientific RepoRts | 5:13969 | DOi: 10.1038/srep13969 Staphylococcus aureus 9 , and BmrR in Bacillus subtilis 10 . These regulators inhibit or stimulate the expression of their target genes to contribute to bacterial drug resistance.
TetR is a large family of transcriptional regulators that contains a conserved helix-turn-helix DNA-binding domain and a C-terminal ligand regulatory domain. These regulators usually serve as repressors and are widely distributed among bacteria 11 . The most frequently characterized function of TetR proteins is regulation of efflux pumps and transporters, which are involved in antibiotic resistance and toxic chemical compound tolerance [12][13][14][15] . For example, the TetR of E. coli controls expression of the gene encoding a tetracycline efflux pump responsible for drug resistance 16 . TetR binds to the promoters of efflux pump genes and is regulated by a plethora of ligands that can cause protein conformational changes and eradicate protein binding, thereby relieving its repression of transcription 17 . In M. tuberculosis, the transcription factor Rv3066, which belongs to the TetR family, was recently found to bind specific co-activator drug molecules (ethidium) and to regulate bacterial drug resistance 18 . Rv3066 can directly bind ethidium and can de-repress the expression of a multidrug transporter operon, mmr 19 . The genomes of both M. tuberculosis and M. bovis BCG encode a large group of TetR family regulators 20,21 . However, transcription factors that can directly bind the first-line anti-TB drugs remain uncharacterized, and the molecular network through which the bacteria respond to the drugs remain largely unclear in M. tuberculosis and related mycobacterial species.
M. bovis BCG is a vaccine strain 21 that has been used as a model strain for studying gene regulatory mechanisms in mycobacterial species, including the pathogenic strain M. tuberculosis. In this study, using M. bovis BCG as a model strain, we screened and characterized InbR, the first INH-binding transcriptional factor that regulates mycobacterial susceptibility to multiple drugs. The results showed that InbR functions as a repressor, and while its overexpression decreased bacterial susceptibility to INH, and its disruption led to supersensitivity of M. bovis BCG to INH. InbR was found to regulate the expression of multiple genes, including the iniBAC operon. Furthermore, we proposed an INH-inducible sequential signal cascade, in which InbR functions as a master regulator and plays an important role in the regulation of mycobacterial susceptibility to multiple anti-TB drugs.

InbR positively regulates INH resistance in M. bovis BCG. Only a few transcription factors have
been reported to contribute to mycobacterial drug resistance to date. To identify potential regulators that contribute to M. tuberculosis INH resistance, we screened a transcriptional factor library by spotting recombinant M. bovis BCG strains, in which the corresponding transcriptional regulator was overexpressed by the constitutive strong promoter hsp60 21 , on plates containing INH (2 μ g/ml). First, all the annotated putative transcriptional regulators (approximately 300 ORFs) of M. tuberculosis were cloned in a batch into the overexpressing plasmid pMV261. Secondly, each recombinant strain was spotted onto 7H10 agar plates that contained 2 μ g/ml INH. As a result, those strains that were more resistant to INH were able to grow and thus were identified as primary candidates. To avoid eventual random mutations that may confer drug resistance, the assays were repeated three times for primary candidates, and finally the drug susceptibility of recombinant strains were attributed to the overexpression of the candidate genes.
A TetR family transcription factor encoded by Rv0275c, designated as InbR, was isolated. As shown in Fig. 1A, we measured the growth of inbR-overexpressing and pMV261 empty vector M. bovis BCG strains on the surface of a solid agar medium with or without INH. When a gradient of different concentrations of mycobacterial strains was spotted on the surface of a solid agar medium without INH, similar bacterial lawns were observed for both the inbR-overexpressing and pMV261 empty vector strains (Fig. 1A, left panel). By contrast, while the same concentration gradient of mycobacterial strains were spotted on a plate containing 2 μ g/ml INH, the bacterial lawn for the pMV261 empty vector BCG strain was smaller than that for the inbR-overexpressing strain, indicating that the strain overexpressing inbR was more resistant to INH than the wild-type strain (Fig. 1A, right panel). This finding suggested that InbR was potentially involved in the regulation of INH-drug resistance in M. bovis BCG.
Orthologs of Rv0275c (InbR) were identified based on sequence similarity and the conservation of adjacent genes. Strikingly, InbR and its orthologs were found to be transcribed divergently from a hypothetical protein (Fig. 1B). The Rv0275c region is highly conserved within M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG (100% amino acid identity over the entire length of the protein), but not in Mycobacterium smegmatis (53% amino acid identity). The gene inbR encodes a 241-residue protein containing a typical TetR_N superfamily domain within an AcrR domain (Fig. 1C), which suggests that InbR belongs to the TetR/AcrR family of transcription factors.
We further assayed the regulatory effect of InbR on the growth of M. bovis BCG in response to INH by determining mycobacterial growth curves. Prior to this assay, the M. bovis BCG inbR-deleted mutant strain (BCG/Δ inbR) was obtained (Fig. S1), together with the complementary strain (BCG/Δ inbR comp). As shown in Fig. 2, no obvious difference was observed in the growth of the pMV261 empty plasmid and inbR-overexpressed BCG strains in 7H9 medium in the absence of drugs ( Fig. 2A, left panel). However, compared with the pMV261 empty plasmid strain, the inbR-overexpressed BCG strain grew significantly better than the pMV261 empty plasmid strain in 7H9 medium containing 1 μ g/ml of INH ( Fig. 2A, right panel; p < 0.05). Without INH, the growth of the inbR-deleted strain (BCG/Δ inbR) and wild type (BCG/WT) have similar growth curves (Fig. 2B, left panel). With 0.1 μ g/ml of INH, the growth of the inbR-deleted strain was significantly inhibited compared with that of the wild type (Fig. 2B, right panel). Additionally, this type of inhibition can be complemented in the complemented strain (Fig. 2C). Moreover, overexpression of inbR decreased the INH susceptibility of the M. tuberculosis H37Ra strain as well (Fig. S2). These results are consistent and indicated that InbR positively regulates INH resistance in M. bovis BCG.
InbR recognizes a palindromic motif and specifically binds to its promoter as an auto-repressor.
In mycobacteria, many TetR family transcriptional factors possess an auto-regulating function. We used an electrophoretic mobility shift assay (EMSA) to examine the binding of the InbR (Rv0275c) protein to the upstream region of its own operon in vitro. As shown in Fig (Fig. 3B, lane 2). By contrast, the pre-immune serum failed to precipitate significant amounts of DNA (Fig. 3B, lane 3). In addition, Rv3430cp, the promoter of an unrelated gene, used as negative control, could not be recovered with InbR antiserum. These findings strongly suggested that InbR could bind with its own promoter region. By using β -galactosidase assays, we further characterized that InbR functions as an auto-repressor (Fig. S3).
We characterized the DNA binding motif of InbR by Dye primer-based DNase I footprinting assays. As shown in Fig. 3C, when increasing amounts of InbR protein (0-2 μ M) were co-incubated with DNaseI, the region around TGCCGCTAATTATGGAAACACCTGTATCCTGATATTGGCCGG was obviously protected on the coding strand. The protected DNA region extended from position − 72 to − 30 in the DNA strand (Fig. 3C). A palindromic motif formed by two inverted repeats partially matched, which was separated from each other by two nucleotides, was found in this region. Further EMSA assays confirmed the significance of the motif for specific recognition by InbR. DNA substrate mutants were synthesized ( Fig. 3C) and EMSA assays were conducted (Fig. 3D). As shown in Fig. 3D (right panel, Lane 6-10), InbR lost the ability to bind with Rv0275cp4 in which the two inverted repeats were replaced by random sequences. By contrast, replacement of either part of the repeat or the interspaced sequence did not abolish their interaction (Fig. 3D, lane 6-15, lane [21][22][23][24][25], although the binding was a little bit weaker compared with that of inherent Rv0275cp1. These results suggested that the binding of InbR may be not very precise and a flexible and partial mismatch is allowed. In conclusion, InbR is an auto-repressor and the auto-regulation of InbR relies on a palindromic sequence motif.  InbR directly binds INH and the binding represses its DNA-binding activity. As far as we know, overexpression of inbR increase INH resistance. On this basis, we further examined whether INH induced the expression of inbR in M. bovis BCG by quantitative RT-PCR (qRT-PCR). M. bovis BCG strains were grown until the logarithmic growth phase (OD 600 = approximately 0.6) and various concentrations of INH (0.5 μ g/ml, 1 μ g/ml, and 2 μ g/ml) were added to the medium. Cells were harvested 24 h later and qRT-PCR was performed. We found that inbR induction was increased by 1.2-fold, 1.67-fold, and 4.08-fold under INH concentrations of 0.5 μ g/ml, 1 μ g/ml, and 2 μ g/ml, respectively (Fig. S4). The results implied that high concentrations of INH will significantly induce the expression of inbR in vivo and interactions between InbR and INH are possibly present.
EMSA assays were subsequently conducted to check the possible interaction between INH and InbR. As shown in Fig Consistently, no response was observed when the same amount of unrelated small molecules, such as guanosine-5′ -triphosphate (GTP) or cyclic diguanylate monophosphate (c-di-GMP), was passed over the His-InbR-immobilized NTA chip (Fig. 4B, right panel). The results showed that InbR directly binds INH.
In addition, SPR experiments were conducted with an immobilized promoter DNA on a chip and InbR in different conditions. InbR promoter DNA was immobilized on the SA chip, and proteins with or without small molecules were passed over. As shown in Fig. 4C, when increasing concentrations of the InbR protein (0.5 μ M to 2 μ M) were passed, corresponding increases in response values were observed (Fig. 4C, left panel). By contrast, unrelated Rv0135c protein did not show any response when passed over the chip. In addition, when InbR was treated by increasing concentrations of INH (20 μ M to 80 μ M INH co-incubated with 0.4 μ M InbR) prior to use, corresponding decreases in response values were observed (Fig. 4C, right panel). By contrast, with identical treatment by an unrelated small molecule GTP, the response value did not change (Fig. 4C, right panel).
These results jointly indicated that InbR binds INH and the binding represses its DNA-binding ability.

The function of InbR is not INH-specific and the mode of action is complicated. Although
InbR does not bind either EMB or RIF, relationships between InbR and the drugs still exist. Minimal inhibitory concentrations (MIC) of wild type, inbR-overexpressing, inbR-deleted and complementary strains were tested with INH, RIF, EMB and mitomycin C (MMC). The MICs of the inbR-deleted strain were all lower compared with those of the wild-type strain (Table 1). By contrast, the MICs of the inbRoverexpressing strain were all higher than that of the wild-type (Table 1). Additionally, growth of the inbR-deleted strain was significantly inhibited by either EMB or RIF at a low concentration in which the wild type strain grew very well (Fig. S6). That is to say, disrupting the inbR gene made the M. bovis BCG strain more sensitive to multiple drugs, whereas overexpressing inbR decreased the susceptibility, and the results suggested that the function of InbR is not INH-specific.
To further elucidate the mechanism by which InbR regulates drug resistance, we performed microarray analyses on inbR-overexpressing and INH-treated wild type M. bovis strains. While comparing the results with that of the non-treated wild type strain, many genes that had consistent expression profiles were identified (Table 2 and Table S5). On the one hand, ribosomal proteins, including S18, L9, S19, L22, S3, L16, L29, S17 and L30, iniBAC and several hypothetical proteins were upregulated in both inbR-overexpressing and INH-treated strains. On the other hand, a large number of metabolic enzymes, including pqqE, lldD1, echA7, gltA1, fadE12, accA2, accD2, gltA1, narG, narH and narJ, and regulatory proteins such as sigI, pfkB, devR were all downregulated. Moreover, there were also many genes with expression profiles that are different in inbR-overexpressing and INH-treated strains. For example, argJ, argB, argD, argE and argR are only upregulated in the former strain. These results suggest InbR uses a complex network to conduct multiple levels of regulation.
We performed qRT-PCR assays to verify the differential expression of several important genes in the inbR-overexpressing and the inbR-deleted strains. On the one hand, expression of Rv0081 and dosR were downregulated (0.3-fold or 0.02-fold) in the inbR-overexpressing strain (Fig. 6A), and upregulated (2.5-fold or 3.8-fold) in the inbR-deleted strain (Fig. 6B). On the other hand, the expression of the groEL1, groEL2 and iniBAC operons was upregulated in the inbR-overexpressing strain (Fig. 6A), and downregulated in the inbR-deleted strain (Fig. 6B). These qRT-PCR results were consistent with the results of the microarray and showed that whenever a gene is a direct or indirect target, there is regulation by InbR.
In summary, InbR regulates bacterial susceptibility to multiple anti-TB drugs in M. bovis BCG, via regulation of a large number of genes.

Discussion
The molecular network through which M. tuberculosis responds to anti-TB drugs and the intrinsic regulatory mechanism underlying mycobacterial INH resistance remain largely unclear. In the present study, we report a TetR family regulator; i.e., InbR, which interacts directly with the first-line anti-TB drug INH in M. bovis BCG. Overexpression of inbR decreased mycobacterial INH susceptibility, whereas disrupting inbR made the mycobacteria supersensitive to multiple anti-TB drugs. Most interestingly, we provide evidence that INH can directly bind to InbR and negatively affects the regulator's DNA-binding ability. Thus, we have uncovered a novel mechanism underlying regulation of mycobacterial susceptibility to INH.
The TetR/AcrR family regulators usually function as repressors and are widely distributed among many bacteria 11 . Most of these proteins are involved in the regulation of drug resistance, biosynthesis of antibiotics, osmotic stress, and bacterial pathogenicity 11 . The AcrR operon of E. coli contains three genes; namely, acrR, acrA, and acrB, the last two of which are multidrug resistant efflux pumps 22,23 . By comparison, InbR has a typical AcrR domain but, unlike in E. coli, is encoded in a single operon. Targets of InbR were, therefore, going to be elucidated. In the present study, we provided evidence to show that InbR acts as an auto-repressor and regulates the expression of a large number of genes. Among these genes, many overlapping genes of InbR regulon genes and INH responsive genes were identified (Table 2  and Table S5). INH responsive genes such as iniBAC, have been shown to be involved in tolerance to multiple anti-TB drugs 5 . Therefore, similar expression profiles for these genes may also give multiple drug resistance to inbR-overexpressing strains. In the InbR regulon, some are direct targets, while the others are indirect targets. Many genes are not drug specific genes in mycobacteria but play roles in multiple stress adaptation. In addition, a ChIP-seq assay revealed that direct targets of InbR are enriched in the GO term small molecule binding. This result implied that the binding of small molecules play an important role in InbR's mode of action. Therefore, other types of small molecules may be preventing targets of InbR regulon genes as well. Additionally, this could be an acceptable explanation for InbR INH-nonspecific functions.
As has been revealed by microarray analysis and qRT-PCR results, InbR could strongly induce the expression of the operon iniBAC (Table 2 and Fig. 6). The iniBAC operon encodes transport-related genes in mycobacteria and confers multiple anti-TB drug tolerance to M. tuberculosis and M. bovis BCG 5 . It is believed that the effect of InbR on multidrug resistance in M. bovis BCG are, mainly or partially, due to the overexpression of the iniBAC operon. Interestingly, a subsequent ChIP-seq assay revealed a high quality peak (qvalue = 1.4E5) downstream of iniBAC. Therefore, the regulation of iniBAC is distinct; for example, by antisense RNA. Moreover, InbR may also regulate iniBAC indirectly. For example, five regulators were reported as regulators for iniBAC in TBDB (http://TBDB.org, Rv0081, Rv0967, Rv1353c, Rv1956 and Rv2250c 37,38 ), in which Rv1956 and Rv1353c were the direct targets of InbR (ChIP-seq peaks found upstream, Table S5), while Rv0081 and Rv0967 were the indirect targets of InbR (down-and upregulated in inbR-overexpressing strain, respectively, Table S5). Although the details were not very clear, it is logical to conclude InbR may regulate iniBAC expression through direct and/or indirect pathways.
One interesting finding is that InbR could regulate susceptibilities of multiple drugs. As we know, drug resistance in M. tuberculosis results primarily from acquisition of chromosomal mutations in genes encoding the drug target proteins, such as katG and inhA [24][25][26] . Nonetheless, gene expression changes were also thought to introduce drug resistance. For example, downregulation of katG was found to be highly associated with isoniazid resistance in M. tuberculosis 27 . Moreover, whiB7 was believed to be one of the main causes of mycobacterial intrinsic drug resistance 28 . In general, the affection for a transcriptional regulator to drug resistance is quite different from an enzymatic gene such as katG. The effect of katG follows a very simple rule: the activation of pro-drug INH. Inactivation of katG leads to defects Continued in INH activation thus introducing drug resistance. By contrast, the effect of a transcriptional regulator would be much more complex. In living cells, regulators set up a network and work jointly, which is flexible and stable. Omitting a single regulator that is not lethal may not affect the function of such a network.
In this study, we found InbR could bind INH and positively regulate drug resistance in mycobacteria. Molecular mechanisms were also investigated and several clues were found; however, the biological role for this novel regulator InbR is still not fully understood and further studies are needed.
In conclusion, this study showed that the TetR-family transcriptional regulator InbR binds isoniazid and influences multidrug resistance in M. bovis BCG.

Experimental Procedures
Strains, plasmids, enzymes and reagents. E. coli BL21 (λ DE3) cells and pET28a were purchased from Novagen (Darmstadt, Germany) and were used to express proteins. Restriction enzymes, T4 ligase, modification enzymes, DNA polymerase, dNTPs, and all antibiotics were obtained from TaKaRa Biotech (Shiga, Japan). PCR primers were synthesized by Invitrogen (Carlsbad, USA). Ni-NTA (Ni 2+nitrilotriacetate) agarose was purchased from Qiagen (Hilden, Germany). 7H9 and 7H10 broths were purchased from Becton, Dickinson Company (New Jersey, USA). Antibodies were obtained from the Wuhan laboratory animal center of CAS (Wuhan, China).  Table 2. Expression patterns of 20 featured gene clusters in inbR-overexpressed and INH induced strains. * log 2 transformed expression values in microarray analysis. ** ChIP-seq peaks identified in inbR overexpressed strain. Up, upstream of the operon or gene; Dn, downstream of the operon/gene; In, inside of a gene; NA, peak is not available. Peaks are visualized in Figure 5 and Figure S7.
with the recombinant plasmid, were grown in 200 ml of LB medium up to OD 600 of 0.6. Protein expression was induced by the addition of 0.3 mM isopropyl β -D-1-thiogalactopyranoside (TaKaRa). Harvested cells were resuspended and sonicated in binding buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; and 10 mM imidazole), and the lysate was centrifuged at 10,000 × g for 30 min. The cleared supernatant was loaded onto the affinity column. The column-bound protein was washed with buffer (20 mM Tris-HCl,  Table 2) that have been listed in Table 2 ChIP-PCR and ChIP-seq assays. Chromatin immunoprecipitation (ChIP) was performed as described previously 29 with modifications. M. bovis BCG cells were grown in 100 ml 7H9 medium, fixed with 1% formaldehyde, and stopped with 0.125 M glycine. Crosslinked cells were harvested and resuspended. The sample was sonicated on ice and the average DNA fragment size was determined to be approximately 0.5 kb. A 100 μ l sample of the extract was saved as the input fraction, whereas the remaining 900 μ l was incubated with 10 μ l of antibodies against corresponding proteins or preimmune serum under rotation for 3 h at 4 °C. The complexes were immunoprecipitated with 20 μ l 50% protein A agarose for 1 h under rotation at 4 °C. The immunocomplex was recovered by centrifugation and resuspension in 100 μ l TE (20 mM Tris-HCl, pH 7.8; 10 mM EDTA; and 0.5% SDS). Crosslinking was reversed for 6 h at 65 °C. The DNA samples of the input and ChIP were purified, resuspended in 50 μ l TE, and analyzed by PCR with Platinum Taq (Invitrogen). The amplification protocol included one denaturation step of 5 min at 95 °C, then 32 cycles of 1 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C. For the ChIP-seq assay, ChIP-enriched DNA was obtained similarly, except that the fragment size was approximately 300 bp, which is the desired size for Illumina short DNA library construction. Sequencing libraries were constructed following the manufacturer's instruction and then subject to Illumina HiSeq2000/2500 instruments (BGI, Shenzhen, China). Short reads were aligned using Bowtie2 30 and peaks were called by MACS 31 . Peaks were annotated using Bioconductor toolbox (http://bioconductor.org).
Dye primer-based DNase I footprinting assay. The DNase I footprinting assay was performed as previously described 32 . A 420-bp fluorescently labeled DNA fragment that encompassed bases − 200 to + 200 of the translational start site of Rv0275c was generated by PCR amplification. The fluorescently labeled probe was subjected to the same binding reaction as in EMSA. Then, 0.0025 U of DNase I was added and incubated for 5 min at room temperature. The digested DNA fragments were purified. The samples were analyzed with the 3730 DNA analyzer coupled with a G5 dye set using an altered default genotyping module that increased the injection time to 30 s and the injection voltage to 3 kV. The 420-bp fragment was sequenced using special primers in the Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit (USB, Inc., Cleveland, OH, USA) following the manufacturer's instructions. Electropherograms were analyzed and aligned using the GENEMAPPER software (version 4.0, Applied Biosystems).

Microarray analysis.
Microarrays used in this study consisted of 15,744 60-mer probes, which were synthesized in situ by Agilent Technologies. The probes were designed based on the genome sequences of M. bovis BCG Pasteur_1173P2_uid58781 (GenBank accession numbers: NC_008769) and covered 3934 ORFs. Each probe was repeated thrice on the array. The inbR-overexpressing M. bovis BCG strain, M. bovis BCG wild-type strain, and INH-treated strain (M. bovis BCG wild-type strain grown on exponential phase OD 600 ≈ 0.8 and treated with 0.5 μ g/ml INH for 24 h) grown on exponential phase OD 600 ≈ 1.2 were harvested. Total RNA was extracted and purified using an RNeasy mini kit (Cat. #74106, QIAGEN, GmBH, Germany) following the manufacturer's instructions. RNA integrity was determined by utilizing RNA integrity number (RIN) generated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat. #5190-2305, Agilent Technologies) following the manufacturer's instructions. Labeled cRNA (complementary RNA) were purified using the RNeasy mini kit.
Each slide was hybridized with 600 ng Cy3-labeled cRNA using a Gene Expression Hybridization Kit of Agilent Technologies (Cat. #5188-5242) according to the manufacturer's instructions. After 17 h of hybridization with 15744 60-mer probes, slides were washed in staining dishes (Cat. #121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat. #5188-5327, Agilent Technologies) following the manufacturer's instructions. Slides were scanned using an Agilent Microarray Scanner (Cat. #G2565CA) with default settings; Dye channel: Green; Scan resolution = 5 μ m; and PMT = 100% and 10%, 16 bit. Data were extracted with Feature Extraction software (ver. 10.7, Agilent Technologies). The raw data were normalized using the Quantile algorithm in the Gene Spring software (ver. 11.0, Agilent Technologies). Normalized microarray expression data deemed significant (P ≤ 0.05) from the InbR-overexpression M. bovis BCG or BCG exposed to INH were selected, and the genes with fold change > 2.0 were selected for further analysis.
Quantitative real-time PCR. Isolation of mRNA and cDNA from mycobacterial strains was performed as described previously 33 . For real-time PCR analysis, gene-specific primers (Table S4)  Surface plasmon resonance (SPR) analysis. SPR analysis was carried out in a Biacore 3000 instrument (GE Healthcare) with nitrilotriacetic acid (NTA) and SA sensor chips as described previously 35,36 . The assays were performed at 25 °C. For the binding of INH with proteins, a His-tagged protein was immobilized onto NTA chips at densities of approximately 1,200 response units (RU). INH was used as the ligand and was diluted in HBS buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 50 μ M EDTA; 5 mM ATP; and 0.005% BIAcore surfactant P20) at concentrations of 8 nM, 40 nM, and 200 nM, and injected at 10 μ l/min for 5 min. GTP was substituted for INH in the negative control. An overlay plot was produced using BIAevaluation 3.1 software to depict the interaction between INH and proteins. To assess the binding of DNA with proteins, biotinylated Rv0275cp probes were immobilized onto streptavidin (SA) chips at densities of approximately 200 (RU). His-tagged Rv0275c, Rv0135c or His-tagged Rv0275c-INH, and Rv0275c-GTP, were diluted in HBS buffer at concentrations of 1, 2, 4, or 4 μ M protein + 0.2, 0.4, and 0.8 μ M INH and injected at 10 μ l/min for 5 min. GTP was substituted for INH in the negative control.
Determination of the MIC of anti-TB drugs. MIC determination was performed as previously described 2 . Briefly, M. bovis BCG/pMV261, inbR-deleted mutant strain and inbR-overexpression strain were grown to OD 600 of 1.0 and diluted to approximately 1 × 10 7 cfu·ml −1 . Then, 0.05 ml of the dilution was used to inoculate 5 ml of Middlebrook 7H9 media containing various concentrations (0-1.28 μ g ml −1 ) of four anti-TB drugs, namely, INH, RIF, EMB, and MMC. The cultures were incubated while shaking at 37 °C for 2 weeks. The MIC was calculated as the concentration of each drug that inhibited bacterial growth.

Determination of mycobacterial growth curves and the effect of antibiotics.
To determine mycobacterial growth curves and the effect of antibiotics, the recombinant strains were grown for a week in Middlebrook 7H9 media (supplemented with 10% albumin dextrose catalase, 0.05% Tween-80, and 0.2% glycerol) containing 30 μ g/ml Kan. Cells were cultured to an OD 600 between 1.5 and 2.0, and each culture was diluted (4:100) in 100 ml of fresh 7H9 broth. The cultures were then allowed to grow further at 37 °C with shaking at 200 rpm. When cells entered a log growth phase (OD 600 of approximately 0.4), the indicated concentration of each antibiotic was added. The cultures were then allowed to grow further at 37 °C with shaking at 120 rpm. Aliquots were obtained at the indicated times, and the cultures were plated on 7H10 medium (supplemented with 0.2% glycerol) to determine colony-forming units 33 .