Method Details

SILAC Cultures were grown to saturation in 7H9+ADS, Hygromycin, Kanamycin, 40 μg/mL Lysine, 240 μM Arginine. Cultures were then diluted to 0.02 in 7H9+ADS, Hygromycin, Kanamycin, and 40 μg/mL Lysine, 240 μM Arginine, or the same concentration of 13C Lysine, 13C Arginine. At OD 600 = 0.5 100 ng/mL ATc was added for 1 hour to induce I-SceI expression. Cells were collected at 3,700xg for 10 minutes at 4°C and resuspended in 8 M Urea, 0.1 M Tris-HCl (pH = 7.9), 150 mM NaCl, 200ul of both phosphatase inhibitors cocktails 2 and 3 and protease inhibitor (0.4ml of 25X cOmplete Protease Inhibitor Cocktail). Cells were lysed using sonication 3 times for 1 minute at 30%. Lysates were then clarified at 4°C for 10 minutes at 20,000xg.

Sample preparation for SILAC Mass spectrometry analysis Disulfide bonds were reduced by incubation with 4 mM TCEP for 30 minutes at room temperature, and free sulfhydryl groups were alkylated by incubation with 20 mM iodoacetamide for 30 minutes at room temperature in the dark. Samples were diluted back to 2 M urea by addition of 0.1 M Tris-HCl (pH = 8.0), and trypsin was added at an enzyme:substrate ratio of 1:100. Lysates were digested overnight at 37°C. Following digestion, the samples were concentrated using SepPak C18 cartridges. The C18 cartridge was washed once with 1 mL of 80% acetonitrile, 0.1% trifluoroacetic acid, followed by a 3 mL wash with 0.1% TFA. 10% TFA was added to each sample to a final concentration of 0.1% after which the samples bound to the cartridge. The cartridge was washed with 3 mL of 0.1% TFA after binding, and the peptides were eluted with 40% ACN, 0.1% TFA. Following elution, the peptides were lyophilized to remove all water and organic solvent. Phosphopeptides were fractionated using hydrophilic interaction chromatography (HILIC). Buffers used for HILIC separation were HILIC buffer A (2% ACN, 0.1% TFA) and HILIC buffer B (98% ACN, 0.1% TFA). Peptides were resuspended in 90% HILIC buffer B and loaded onto a TSKgel amide-80 column. Peptides were separated at a flow rate of 0.5 mL/min using a gradient from 90% to 85% HILIC buffer B for 5 minutes, 85% to 55% HILIC buffer B for 80 minutes, then 55% to 0% HILIC buffer B for 5 minutes. Fractions were collected every 2 minutes and the 22 fractions previously determined to contain the majority of phosphopeptides were lyophilized. Following HILIC fractionation, fractions were further enriched for phosphopeptides using titanium dioxide magnetic beads using the manufacturer’s protocol. Following titanium dioxide enrichment, sample were lyophilized and resuspended in 0.1% formic acid for mass spectrometry analysis.

Mass spectrometry analysis of RecA phosphosite Cells were collected and washed twice with ice cold phosphate-buffered saline (155 mM NaCl, 3 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , pH = 7.4, PBS) and frozen in liquid N 2 . For lysis, bacterial cells were thawed on ice and lysed in lysis buffer (8 M urea, 25 mM Tris-HCl, 150 mM NaCl, phosphatase inhibitor 2 and 3 and protease inhibitors. Lysates were sonicated three times at 30 – 40% power for 15 s each with intermittent cooling on ice, followed by centrifugation at 14,000 rpm for 30 min at 4°C. The supernatants were transferred to a new tube and the protein concentration was determined using a BCA assay. For each time point, 5 mg of total protein was used. Protein was reduced with 5 mM DTT at 56°C for 30 min, cooled to room temperature, and alkylated with 11 mM iodoacetamide in the dark at room temperature for 30 min. The alkylation was then quenched by the addition of an additional 5 mM DTT. Samples were diluted 6-fold with 50 mM NH 4 HCO 3 and digested overnight with trypsin (1:50) at 37°C. The next day, the digestion was stopped by the addition of 0.25% TFA (final v/v) and centrifuged at 10,000 rpm for 10 min at room temperature to pellet precipitated lipids and collected the cleared supernatant. Supernatant were desalted on a SepPak C18 cartridge 500 mg. Desalted sample (1%) was directly injected to LC-MS/MS to quantify unmodified RecA peptides and remaining 99% of sample was lyophilized for phosphopeptides enrichment.

RecA phosphopeptides enrichment Adachi et al., 2016 Adachi J.

Hashiguchi K.

Nagano M.

Sato M.

Sato A.

Fukamizu K.

Ishihama Y.

Tomonaga T. Improved proteome and phosphoproteome analysis on a cation exchanger by a combined acid and salt gradient. Kettenbach and Gerber, 2011 Kettenbach A.N.

Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Phosphopeptide enrichment was performed as described (), with minor modifications. 5 mg of lyophilized peptides were resuspended in 500 μL of binding buffer (2 M lactic acid in 50% ACN). Resuspended peptides were incubated with 20 mg of titanium dioxide microspheres for one hour, by a thermostat vortex mixer on the highest speed setting at room temperature. Afterward, the beads were washed twice with 500 μL of the binding solution and three times with 500 μL (50% ACN / 0.1% TFA), and phosphopeptides were eluted sequentially with 60 μL of 5% ammonium hydroxide, 5% piperdine and 5% pyrrolidine solution. Peptide elutions were combined and quenched with 120 μL of 50% ACN / 5% formic acid and were dried by speedvac. Enriched phosphopeptides were further fractionated in seven fractions with stepwise elution by TFA and ammonium acetate using SDB-SCX Stage-Tip (). The elution buffer contained 0.5% TFA, 30% ACN for fraction 1; 1% TFA, 30% ACN for fraction 2; 2% TFA, 30% ACN for fraction 3; 3% TFA, 30% ACN for fraction 4; 3% TFA, 30% ACN, 100 mM ammonium acetate for fraction 5; 3% TFA, 30% ACN, 500 mM ammonium acetate for fraction 6; 30% ACN, 100 mM ammonium acetate for fraction 7.

LC-MS/MS analysis Desalted peptides and fractionated phosphopeptides were resuspended in 10 μL of 3% ACN/0.1% formic acid and were injected onto a C18 capillary column on a nano ACQUITY UPLC system (Water) which was coupled to the Q Exactive plus mass spectrometer. Peptides were eluted with a non-linear 110 min gradient of 0.5 – 50% buffer B (0.1% (v/v) formic acid, 100% ACN) at a flow rate of 300 nL/min. After each gradient, the column was washed with 90% buffer B for 5 min and re-equilibrated with 99.5% buffer A (0.1% formic acid, 100% HPLC-grade water). MS data were acquired with combined two scan events corresponding full scan and a Parallel Reaction Monitoring (PRM) method targeting the eight RecA peptides. Target value for the full scan MS spectra was 1 × 106 ions in the 380-2000 m/z range with a maximum injection time of 30 ms and resolution of 70,000 at 200 m/z with data collected in profile mode. The PRM method employed at resolution of 17,000 at 200 m/z, a target AGC value 2 × 105 and maximum fill times of 100 ms. The precursors ions of each targeted peptide were isolated using a 1.5 m/z unit window and fragmented by higher-energy C-trap dissociation with normalized collision energy of 27 eV.

Data processing Data analysis was performed using Xcalibur (version: 4.0.27.19, Thermo Fisher Scientific) and the area under the curve of selected intense fragment ions summed to determine the quantity of the respective peptide.

DNA Damage assays M. smegmatis strains were grown in LB with 0.5% glycerol, 0.05% tween80, 0.5% dextrose, 20 μg/ml streptomycin until they reached saturation. M. tuberculosis strains were grown in 7H9 medium + OADC + 20 μg/mL Kanamycin to saturation. Cultures were diluted to an OD 600 = 0.02 (M. smegmatis) or 0.1 (M. tuberculosis) and grown to OD 600 = 0.6, washed once with an PBS + 0.05% tween80 and serially diluted onto 7H10 agar plates. Agar plates were treated with various doses of UV radiation with a Stratagene UV stratalinker 1800. Plates were wrapped in foil (to prevent potential effects of photolyase) and incubated at 37°C. For treatment with the clastogen methyl methanesulfonate (MMS), M. tuberculosis was grown as indicated above to an OD 600 of 0.5 and treated with PBS or 0.2% MMS (final) for 1 hour. Cultures were washed with PBS + 0.05% tween80 three times to remove MMS and then diluted to an OD 600 = 0.03. Growth was measured by OD 600 for 10 days. For ionizing radiation exposure, cells were grown to saturation in 10 mL of LB media and then diluted in fresh media. When cultures reached OD 600 ∼0.3, they were centrifuged and resuspended in 5 mL of PBS with 0.05% tween80. 100 μL aliquots were treated with IR from a 137Cs source that delivers 10 Gray/min on a rotating platform to ensure uniform exposure. Pooled aliquots were used for plating dilutions. Ciprofloxacin exposure was at 10 μg/mL for 1 or 3 hours.

Western Blot Lysates were prepared from cells at OD600 of 0.6 and protein quantities were roughly normalized to equivalent cell number. Equal loading was confirmed with RpoB antibodies as a loading control. Commercially available anti-RecA (Abcam 63797) was used at a 1:3,000 dilution and anti-RpoB was used at a 1:20,000 dilution, both incubated for 1 hour. Anti-RecA antisera were raised in rabbits against purified full-length M. smegmatis RecA.

RecA and LexA protein purification The following buffers were used: buffer A (binding): 20 mM Tris-HCl (pH = 8.0), 200 mM NaCl, 50 mM imidazole, 10% glycerol; buffer B (wash): 20 mM Tris-HCl (pH = 8.0), 2 M KCl; buffer C (elution): 20 mM Tris-HCl (pH = 8.0), 0.2 M NaCl, 0.5 M imidazole, 10% glycerol; buffer D (dialysis): 20 mM Tris-HCl (pH = 8.0), 0.2 M NaCl, 10% glycerol; buffer E (lipid vesicle buffer): 20 mM Tris-HCl (pH = 8.0). M. smegmatis recA WT, S207A, S207E S207D, S207N, and LexA were cloned into the pET-Sumo protein expression vector, containing a N-terminal polyhistidine (His 6 ) tag, SUMO, and a Ulp1 cleavage site. DNA sequencing confirmed the full gene was present in each instance without mutations. T7-based expression in ER2566 or BL21-CodonPlus (DE3) expression strain of E. coli. was induced with 0.3 mM isopropyl-β-d-thiogalactoside (IPTG) for 4 hours at 37°C. Cells were collected by centrifugation and resuspended in buffer A and lysed by sonication. Lysate was centrifuged at 20,000 x g at 4°C for 30 minutes. Supernatant was passed over a Ni-NTA or Talon column for immobilized metal affinity chromatography purification, washed with 10 column volumes of buffer A, then buffer B to remove DNA. Protein was eluted with buffer C. Eluted protein was dialyzed overnight in buffer D with ∼0.3 mg of His 6 -labeled Ulp1 protease, which was expressed recombinantly in E. coli and purified. After cleavage protein was run over Ni-NTA column and the flow through was collected containing purified RecA or LexA protein. Isolated proteins in buffer D were concentrated by ultrafiltration (molecular weight cutoff: 10 kDa) to less than 0.5 mL and directly frozen. Purified RecA WT, S207A, S207E, S207D, and S207N proteins were analyzed by reducing SDS-PAGE, and protein band identities and mutant L2 amino acid substitutions were confirmed by in-gel tryptic digestion and subsequent LC-MS/MS analysis.

Strand exchange assay A reaction mixture containing Tris-HCl (70 mM, pH = 7.6), MgCl 2 (10 mM), dithiothreitol (5 mM), ATPγS (2.0 mM), RecA (5.0 μM), 100 nM HPLC-purified 5′ NHS ester Alexa Fluor 488-labeled oligo (IDT), with the following sequence: /5Alex488N/GA AAA TTC GAC CTA TCC TTG CGC AGC TCG AGA AGC TCT TAC TTT GCG ACC TTT CGC CAT C (referred to as 488N-ϕX174-oligo), was incubated at 37°C for 10 minutes to form RecA-ssDNA filaments. D loop formation was initiated with the addition of 1 μg ϕX174 RF1 DNA to the mixture, and incubated at 37°C. The reaction was stopped via addition of Proteinase K and 1% SDS at room temperature. Reaction products were resolved on a 1% agarose gel and imaged on a Typhoon Trio Variable Mode Imager in the green channel (PMT 600, laser 532 nm, normal sensitivity, 100 μm2 pixel size).

ATPase assay The ATPase assay was performed in a 10 μL reaction volume containing 70 mM Tris-HCl (pH 7.6), 5 mM DTT, 10 mM MgCl 2 , and 10 μM ϕX174-oligo + 3 μM RecA. The reaction was initiated by the addition of 2 mM [γ-32P] ATP and incubated for different time intervals (0,10,20,30,40,50,60 min) at 37°C. The reaction was stopped by the addition of 25 mM EDTA. Aliquots (2 μL) were transferred onto polyethylenimine cellulose F sheets and developed with a solution containing 0.5 M LiCl and 1 M formic acid. The TLC sheets were air-dried and the bands were visualized using a Typhoon Trio Variable Mode Imager. The band intensities were quantified using ImageJ and plotted using GraphPad prism (version 7.0). To monitor the effect of lipid vesicles, the reaction mixtures was incubated either in the presence of 100% DOPG vesicles or DOPG:CL vesicles. The reaction was initiated by the addition of 2 mM [γ-32P] ATP and incubated for 30 minutes. The reaction products were resolved by thin layer chromatography (TLC) on polyethylenimine (PEI) cellulose F plates (Merck Millipore).

LexA cleavage assay The LexA cleavage assay was performed in a 10 μL reaction volume containing Tris-HCl (70 mM, pH = 7.6), MgCl 2 (10 mM), dithiothreitol (5 mM), ATPγS (3 mM), RecA (3 μM), 10 μM ssDNA with the following sequence GAT GGC GAA AGG TCG CAA AGT AAG AGC TTC TCG AGC TGC GCA AGG ATA GGT CGA ATT TTC. The reaction mixtures were incubated at 37°C for 15 min. Subsequently, 10 μM LexA was added and further incubated for different time intervals (0, 5, 10, 15, 20 and 25 min) at 37°C. Samples were separated by SDS–PAGE followed by staining with Coomassie Blue. The band intensities were quantified using ImageJ and plotted using GraphPad prism (version 7.0). To monitor the effect of lipid vesicles, the reaction was incubated in the presence of 100% DOPG or DOPG:CL vesicles, Tris-HCl (70 mM, pH = 7.6), MgCl 2 (10 mM), dithiothreitol (5 mM), ATPγS (3 mM), RecA (3 μM), 10 μM oligodT 30 ssDNA. The reaction was incubated at 37°C for 15 minutes and 5 μM LexA was added and further incubated for different time intervals (0, 5, 10, 15, 20 and 25 min) at 37°C. The reaction was terminated by boiling in SDS sample buffer. Samples were separated by SDS–PAGE followed by staining with Coomassie Blue. The band intensities were quantified using ImageJ and plotted using GraphPad prism (version 7.0).

Electrophoretic mobility shift (EMSA) assay Reactions contained Tris-HCI (70 mM, pH = 7.6), MgCl 2 (10 mM), dithiothreitol (5 mM), ATPγS (2.0 mM), RecA (variable concentrations), and 488N-ϕX174-oligo chemically labeled with Alexa Fluor 488 (250 nM) were combined to a final volume of 10 μL and incubated at 37°C for 30 minutes to form RecA-ssDNA filaments. The reaction products were resolved on a 1% agarose gel and imaged on a Typhoon Trio Variable Mode Imager in the green channel (vide supra).

UV induced mutagenesis 10 mL of each strain at OD 600 = 0.6 was transferred to Omnitray single-well plates and exposed to 20 mJ/cm2 UV radiation using a Stratagene UV stratalinker 1800. From each treated sample and its untreated control, 5 mL of culture was transferred to 5 mL of fresh media and shaken at 37°C /150 RPM for 3 hours. From each sample, 1 mL of culture was cultured in duplicate on 7H10 agar plates containing 0.5% glycerol, 0.5% dextrose, and 40 μg/mL rifampicin and incubated at 37°C for 72 hours to determine rifampicin-resistant CFU. Additional duplicates were taken from each sample and dilution-plated on 7H10 agar containing no antibiotic to determine viable CFU. Resistant mutants were then normalized to viable CFU for each set of samples, 10 replicates for each strain. Graph represents average rifampicin-resistant mutants per viable CFU.

Lipid vesicle preparation 100-nm diameter unilamellar vesicles (large unilamellar vesicles) were made from mixtures of lipids by extrusion. Lipid ratios were determined by mass. The lipids dissolved in organic solvent were mixed proportionally according to their mass, dried as a thin film under N 2 , and evacuated via lyophilizing overnight. The lipid mixtures were re-suspended in buffer E, sonicated, and vortexed. Each sample underwent at least two freeze-thaw cycles at −80°C and 37°C. This mixture was extruded with an Avanti Mini-Extruder Set at least 10 times through two stacked 100-nm filters, yielding homogeneous batches of unilamellar vesicles. The size of the vesicles was confirmed by dynamic light scattering, and net charge (Zeta potential) was measured.

RT-qPCR Cultures with 1.25 μg/ml Ciprofloxacin were shaken at 37°C /150RPM for 3 hours. 10 mL samples were collected from each culture in duplicate for RNA preparation. 500 ng of RNA was used to make cDNA using the Thermo Maxima First Strand cDNA synthesis kit for RT-qPCR with dsDNase. RT-qPCR was performed using TaqMan Assay with SigA as the housekeeping gene and comparing the ΔΔCT for each strain treated for 3 hours to its respective untreated control. 600 = 0.6 at 37°C. For each strain, 8 mL of culture was transferred to Extra-Depth disposable Petri dish and exposed to ∼20mJ/cm2 UV radiation using a Stratagene UV stratalinker. From each treated sample and untreated controls 5 mL of culture were transferred to a 30 mL bottle containing 5 mL of fresh media and shaken at 37°C/150 RPM for ∼24 hours. 5 mL samples of untreated culture from each strain were also transferred to 5 mL of fresh media and shaken at 37°C/150 RPM for ∼24 hours. Cell pellets were lysed in TRIzol reagent by bead beating 3 times for 45 s and processed using the Direct-zol Miniprep Plus kit. RNA was treated following the rigorous DNase treatment of the TURBO DNA-free kit. cDNA synthesis and RT-qPCR were similar to protocol used for M. smegmatis cells. Primers for RT-qPCR: M.smegmatis dnaE2 forward primer: GCACTGGCACATCCTCAC

M.smegmatis dnaE2 reverse primer: CCGAACCGGTCCACTAGAT

M.smegmatis dnaE2 TaqMan probe: ACATGGCCTTTGCGGCATCC

M.tuberculosis dnaE2 forward primer: GTCCTGCAATGGGACAAAGA

M.tuberculosis dnaE2 reverse primer: CACCAGGTCTTTCGCATAGT

M.tuberculosis dnaE2 TaqMan probe: CGGCAATCGGCTTGGTGAAATTCG M. tuberculosis strains were grown in 7H9 medium+OADC to OD= 0.6 at 37°C. For each strain, 8 mL of culture was transferred to Extra-Depth disposable Petri dish and exposed to ∼20mJ/cmUV radiation using a Stratagene UV stratalinker. From each treated sample and untreated controls 5 mL of culture were transferred to a 30 mL bottle containing 5 mL of fresh media and shaken at 37°C/150 RPM for ∼24 hours. 5 mL samples of untreated culture from each strain were also transferred to 5 mL of fresh media and shaken at 37°C/150 RPM for ∼24 hours. Cell pellets were lysed in TRIzol reagent by bead beating 3 times for 45 s and processed using the Direct-zol Miniprep Plus kit. RNA was treated following the rigorous DNase treatment of the TURBO DNA-free kit. cDNA synthesis and RT-qPCR were similar to protocol used for M. smegmatis cells. Primers for RT-qPCR:

Cell fractionation Stone, 1974 Stone A.B. A simplified method for preparing sucrose gradients. The indicated strains were exposed to ciprofloxacin as detailed under RT-qPCR. Cells were washed twice with 50 mM HEPES (pH = 7.4) and snap frozen on dry ice, resuspended to 20 mL of 50 mM HEPES (pH = 7.4), and lysed three times via French press at 14,000 psi. 9.5ml of the lysates were spin concentrated at 3,7 00G for 15mins two times in an Amicon Ultra −15 Centrifugal unit (Ultracel −10K) and then 600ul of the concentrated lysate was overlaid on a continuous sucrose gradient of 20 – 60% sucrose in 25 mM HEPES (pH = 7.4), prepared according to the method of Stone () by overlaying a 5.5 mL solution of 60% sucrose in 25 mM HEPES (pH = 7.4) with 5.5 mL of 20% sucrose in 25 mM HEPES (pH = 7.4) in 12 mL Polyallomer ultracentrifuge tubes (Denville). The tubes were then laid flat for 4 hours at room temperature before being returned to an upright position and placed at 4°C overnight followed by centrifugation at 218,000G in a SW40Ti rotor for 6 hours. 1 mL fractions were collected from the bottom of the tube and analyzed by western blotting using affinity purified anti-FtsY at a concentration of 1:2000 and anti-RpoB at 1:10,000 dilution. The membranes were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific), rinsed with 1x PBS, and probed with rabbit anti-RecA at a concentration of 1:5,000.