2.1 Strains and culture conditions M. smegmatis was cultured either in 7H9 liquid medium (BD, USA) supplemented with 0.2% glycerol as carbon source and 0.05% Tween 80 to prevent agglomeration or on 7H10 solid medium (BD, USA) with 0.5% glycerol. Escherichia coli DH5α and BL21 for DNA cloning and protein expression were grown in Luria-Bertani medium. Antibiotics are used for resistance screening, according to the need, 100 µg/ml ampicillin, 100 µg/ml hygromycin, 50 µg/ml kanamycin (for E. coli DH5α and BL21) or 20 µg/ml kanamycin (for M. smegmatis) was added.
2.2 Construction of knockout strains According to Yan Meiyi’s method (Yan et al., 2017), the homologous recombination-mediated deletion was used to construct all M. smegmatis knockout strains. For hxlR knockout strain, the workflow was as follows. The upstream homologous arm and the downstream homologous arm of the hxlR gene were separately amplified by PCR using the genome of M. smegmatis genome DNA as the template and using primer pairs P1/P2 and P3/P4(for primer sequences, see Supplementary Table S1, respectively. cr-hxlR-F and cr-hxlR-R were used for pcr-Hyg plasmids construction. The homology arms and the pcr-Hyg plasmids were then electroporated into the competent cells of M. smegmatis containing pJV53-cpf1, transformants were plated onto 7H10 solid medium containing hygromycin, kanamycin and anhydrous tetracycline (50 ng/ml) and culture at 37℃ for 5 days, single colonies were picked and hxlR knockouts were validated via colony PCR.
2.3 in vitro growth curve assays of M. smegmatis and survival under various stresses The strains were grown to logarithmic growth phase. The bacterial were collected and washed, resuspended in 7H9 liquid medium and diluted to an OD600 of 0.8, 1% inoculum size strains were inoculated into 100 ml of 7H9 liquid medium containing 1% Acetamide (Aladdin, Shanghai, China), then cultured at 37℃ with 180 rpm shaking. OD600 was determined at indicated time points. M. smegmatis was cultured to the logarithmic phase and collected, followed by washing with 1ⅹPBS, resuspended in 7H9 liquid medium and diluted to an OD600 of 0.7-0.8. 2 ml from a stock solution of M. smegmatis were used for the formaldehyde bactericidal assay for 3 days, or diluted to an OD600 of 0.4-0.45, 2 ml from a stock solution of M. smegmatis were used for the H2O2 bactericidal assay for 1 day. Samples were harvested at indicated time points for colony counting.
2.4 Electrophoretic mobility shift assay (EMSA) Promoter regions of hxlR, MSMEG_6581, MSMEG_0198 were amplified from M. smegmatis genome using primers. Various micromole of the His-HxlR protein were pre-mixed with 300-400 ng DNA and 1ⅹEMSA buffer (20 mM HEPES, 500 mM NaCl, 4 mM DTT, 2% glycerol, 2 mM MgCl2, pH = 8.0) and then incubated at 37℃ for 30 min. As needed, the samples were supplemented with or without different concentrations of oxide/reductant dithiothreitol (DTT). The electrophoresis was performed in a 6% non-denaturing PAGE and carried out in a running buffer containing 0.5ⅹTBE buffer at 4℃ for 1 h 30 min. The gel was stained with GoldView and visualized.
2.5 RNA isolation and qRT-PCR analysis Expression of related genes was tested using qRT-PCR. The WT M. smegmatis and ΔhxlR strains were cultured at OD600 = 0.8 in 7H9 liquid media and H2O2 at a final concentration of 1 mM were added and incubated for 3 h. Total RNA was extracted using TRIzol Reagent and purification kit (Tiangen, Beijing, China) according to the manufacture’s protocol. cDNA was synthesized with a high-capacity cDNA reverse transcription kit (Roche, USA). The qRT-PCR expression analysis was performed using CFX96 Real-time PCR Detection System (Bio-Rad, USA). The sigA signal was used as an internal reference gene.
2.6 Cloning, expression and purification of recombinant protein and mutant proteins According to the published procedure, the recombinant proteins were expressed and purified in E. coli (Lin et al., 2017). The upstream and reverse primers for hxlR were used to obtain the hxlR gene fragment and ligation to the pET28a (with SUMO tag, 15.6kDa) plasmid.
2.7 Detection of promoter activity Chromogenic qualitative method: WT, ΔhxlR were grown in 7H9 liquid medium to an OD600 of 0.6-0.8, bacteria solution was plated onto 7H10 solid medium with X-gal (40 µg/ml) and kanamycin (20 µg/ml) at 37°C for 1-2 days, each sample was repeated three times. Quantitative experiments: 100 µl bacterial solution samples were mixed with 900 µl Z-buffer, 20 µL 0.1% (m/v) SDS, 20 µL chloroform and then vortexed for 1 min. Then incubated at 37°C for 30 min to lyse cells. After that, 200 µl ONPG (4 mg/ml) was added and incubated for 1 h, then stopped the reaction with 200 µL Na2CO3 (2.5 mol/L). The samples were centrifuged and added to a 96-well plate. The absorbance was detected at wavelength 420 nm with a microplate reader. Calculation formula=(1000 OD420)/(V T OD600), V represents the volume of the bacterial solution, and T represents the incubation time.