In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation

Culture media, growth conditions, and chemicals.Lysogeny broth (LB; Difco) (35) was used as rich medium for Bacillus subtilis strains. Spizizen minimal medium supplemented with tryptophan (20 μg/ml) was used to grow B. subtilis under defined conditions (36). Nutrient broth (NB; Difco) supplemented with NaCl (85 mM) was used as rich medium for Salmonella enterica strains. No-carbon E (NCE) minimal medium supplemented with trace minerals (37), l-methionine (100 μM), and magnesium sulfate (1 mM) was used to grow S. enterica strains (38). Glycerol (22 mM) was used as the sole carbon and energy source in all cases. All strains were grown at 37°C with shaking (180 rpm). When added to the medium, antibiotics were present at the following concentrations: ampicillin (Fisher Scientific), 100 μg ml⁻¹; and erythromycin (Sigma-Aldrich), 1 μg ml⁻¹. HEPES buffer, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), isopropyl-β-d-1-thiogalactopyranoside (IPTG), and dithiothreitol (DTT) were purchased from Gold BioTechnology; streptothricin sulfate was purchased from G Biosciences.

Bacterial strains.B. subtilis strains used in these studies were derivatives of B. subtilis subsp. subtilis strain 168. Strains JE9142 (trpC2) and JE20317 (trpC2 satA1::erm⁺) were purchased from the Bacillus Genetic Stock Center (Columbus, OH). Other B. subtilis strains were constructed during the course of this work. S. enterica strains were derivatives of S. enterica subsp. enterica serovar Typhimurium strain LT2 and were constructed during the course of this work. All strains and plasmids used are listed in Table 2.

Plasmid construction.All primers used in this study were synthesized by Integrated DNA Technologies (Coralville, IA) and are listed in Table 3. The satA (yyaR) gene was amplified from B. subtilis subsp. subtilis 168 genomic DNA using Phusion polymerase (T_A 57°C) following manufacturer's instructions, and cloned into vectors using the BspQI (New England BioLabs) cloning method (39). The satA⁺ allele was cloned into both pCV1 (40), an l-(+)-arabinose-inducible complementation vector, and pTEV16 (40), an IPTG-inducible overproduction vector that fused a H₆ tag to the N terminus of the protein. For the B. subtilis integration vector pBS1ClacZ (41), the satA⁺ allele and 265 bp upstream of it were amplified from B. subtilis 168 genomic DNA and cloned into the EcoRI and SpeI (ThermoFisher Scientific) restriction sites of pBS1ClacZ. The B. anthracis Ames satA (formerly GFG-5714) gene was codon-optimized for E. coli and synthesized by GenScript Biotech Corporation (China) into the EcoRI and SmaI sites of plasmid pTEV5 (42), an IPTG-inducible overproduction vector that fused a H₆ tag to the N terminus of the protein. For cloning into pCV1, the B. anthracis satA gene was amplified from pBaSAT2 and cloned using the BspQI cloning method as described above. The sequences of vectors were verified (Georgia Genomics Facility, University of Georgia, Athens, GA).

Growth analyses.S. enterica strains were grown at 37°C as described above and challenged with streptothricin (10 μM). B. subtilis strains were also grown at 37°C in Spizizen glycerol minimal medium (36) and also challenged with 10 μM streptothricin.

All growth analyses were performed in 96-well microtiter dishes, with each strain grown under identical conditions in triplicate. Each well contained 198 μl of minimal medium inoculated with 1% (vol/vol) of an overnight culture for S. enterica strains, or 3% (vol/vol) of an overnight culture for B. subtilis strains. All starter cultures were grown for 14 to 16 h on rich medium at 37°C in an innova43 (New Brunswick Scientific) gyratory incubator shaking at 180 rpm. Cell density was monitored at 630 nm using a computer-controlled BioTek ELx808 absorbance plate reader (BioTek Instruments). Readings were acquired every 30 min with continuous shaking. Data were analyzed using the GraphPad Prism 4 software package (GraphPad Software).

Purification of His₆-tagged B. subtilis SatA protein.H₆-BsSatA was overproduced in E. coli strain C41(λDE3) cells (43) carrying plasmid pBsSATA2 (satA⁺). Cells were grown in 1.5 liters of Terrific Broth (Cold Spring Harbor Laboratory protocols) at 37°C to an optical density at 600 nm (OD₆₀₀) of 0.3 to 0.4. At this cell density, isopropyl-β-d-1-thiogalactopyranoside (IPTG; Sigma) was added to a final concentration of 500 μM and cultures were incubated overnight at 15°C in a 2.8-liter flask in an Innova 44 (New Brunswick Scientific) gyratory shaker at 160 rpm. Cells were harvested by centrifugation at 6,000 × g for 15 min, and the cell paste was frozen at −80°C until use. Cell paste was resuspended in binding buffer containing HEPES buffer (50 mM, pH 7.5 at 4°C) containing NaCl (500 mM) and imidazole (20 mM), 1 μg ml⁻¹ lysozyme, 25 μg ml⁻¹ DNase, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Fisher Scientific). Cells were broken by sonication with a 550 sonic dismembrator (Fisher Scientific) for 4 cycles at an amplitude setting of 60 for 30 s on ice for 2 s on, followed by 2 s off. Cellular debris was removed by centrifugation at 40,000 × g for 45 min using an Avanti J-25I floor centrifuge with a JA-25.25 rotor (Beckman Coulter).

The clarified cell extract was loaded onto a 5-ml nickel-nitrilotriacetic acid (Ni-NTA) affinity column (HisPur; ThermoFisher Scientific) and purified as described elsewhere (23). Briefly, the column was washed with 10 bed volumes of binding buffer (HEPES, 50 mM, pH 7.5) containing NaCl (500 mM) and imidazole (20 mM), followed by six bed volumes of wash buffer (HEPES 50 mM, pH 7.5) containing NaCl (500 mM) and imidazole (40 mM). Protein was eluted off the column with six bed volumes of elution buffer (HEPES 50 mM, pH 7.5) containing NaCl (500 mM) and a high imidazole concentration (500 mM). Fractions of eluted protein were pooled and dialyzed against HEPES buffer (50 mM, pH 7.5 at 4°C) with decreasing amounts of NaCl down to 150 mM. Purified protein was flash frozen in liquid nitrogen and stored at −80°C until use. The protein was 81% homogeneous (Fig. S4).

In vitro acetylation assay and thin-layer chromatography.Reaction mixtures (25 μl) containing HEPES buffer (50 mM, pH 7 at 24°C), TCEP (1 mM), [1-¹⁴C]-AcCoA (20 μM; specific radioactivity = 56.8 mCi/mmol), BsSatA (3 μM; extinction coefficient = 4.58 × 10⁴ liter · mol⁻¹ · cm⁻¹ obtained through http://web.expasy.org/protparam/), and streptothricin (150 μM) were incubated at 37°C for 1 h. Reaction mixtures lacking enzyme or substrate were used as controls. Samples from reaction mixtures were spotted onto a Whatman PE SIL G/UV silica gel thin-layer chromatography (TLC) plate (Whatman Ltd.), and developed in a preequilibrated chamber with a mobile phase of 15% (wt/vol) KH₂PO₄. TLC plates were developed for 1 to 2 h, dried, and radioactivity distribution was visualized using a phosphor imager after 16 h of exposure. A Typhoon Trio+ Variable Mode imager (GE Health Life Sciences) with ImageQuant v5.2 software was used to image the phosphor screen.

Streptothricin resistance bioassay.The B. subtilis satA⁺ strain (JE9142) was grown overnight at 37°C in rich medium for 14 to 16 h. A sample (100 μl) of starter culture was added to 3 ml of soft agar and poured onto a rich medium plate. Reaction mixtures containing HEPES buffer (50 mM, pH 7), TCEP (1 mM), AcCoA (400 μM), and various concentrations of streptothricin (5 to 200 μM) were incubated at 37°C for 1 h with or without BsSatA (3 μM). A sample (5 μl) of each reaction mixture was spotted onto satA⁺ (JE9142)-containing plates, which were incubated overnight at 37°C. Sensitivity to streptothricin was determined by the diameter of zones of inhibition. As a negative control, BsSatA was heated to 95°C for 5 min and added to a reaction mixture containing streptothricin (50 μM).

Gel permeation FPLC analysis.H₆-BsSatA protein (125 μg) was injected onto a Superose 12 10/300 gel filtration column connected to an ÄKTApurifier fast protein liquid chromatography (FPLC) system. The column was equilibrated with HEPES buffer (50 mM, pH 7.5) containing NaCl (150 mM). A mixture of standards was applied to the column to generate a calibration curve. A flow rate of 0.5 ml · min⁻¹ was used to develop the column, and elution was monitored at 280 nm. Data analysis was performed using UNICORN 4.11 software (GE Healthcare Life Sciences).

Determination of kinetic parameters.The kinetic parameters for the BsSatA reaction were determined using a continuous spectrophotometric assay that employed 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent) to measure the release of coenzyme A (CoA) from AcCoA at 412 nm (32–34). Briefly, reaction mixtures (100 μl) contained HEPES (50 mM, pH 7), DTNB (0.3 mM), and H₆-BsSatA (0.5 μg), saturating conditions on one of the substrates and varying concentrations of the second substrate. Reaction mixtures containing H₆-BsSatA and AcCoA but no streptothricin were used as control to correct for background. When streptothricin was added to the mixture, AcCoA levels were kept at 100 μM and streptothricin was varied from 100 nM to 2.5 μM. When the concentration of AcCoA varied, the streptothricin level was kept at 10 μM, and acetyl-CoA was varied from 25 μM to 200 μM.

Reaction mixtures were incubated at 25°C and readings were taken every 5 s over 5 min using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Reactions were performed in technical triplicates and biological duplicates. The molar extinction coefficient used for the concentration of the TNB^2- anion was 14,150 M^{−1 ·} cm⁻¹ (Sigma-Aldrich). The initial velocity versus substrate concentration was graphed using Prism 6 (GraphPad) software. Data were fitted to the Michaelis-Menten equation to determine the apparent K_m and k_cat for BsSatA. Catalytic efficiency (k_cat/K_m) was also calculated.

To determine the level of acetylation of streptothricin, reaction mixtures were prepared as described above with AcCoA (500 μM) and various streptothricin concentrations. Reaction mixtures were incubated for 1 h and the final absorbance at 412 nm was used to determine the amount of AcCoA consumed as a function of the concentration of streptothricin added to the reaction mixtures.

Isothermal titration calorimetry.H₆-BsSatA was dialyzed extensively against HEPES buffer (25 mM, pH 7.5; 150 mM NaCl), and binding assays were performed using a Nano ITC isothermal titration calorimeter (TA Instruments). AcCoA or streptothricin was used as titrant; these compounds were solubilized in the final protein dialysate. Protein was present at 40 μM in the sample cell and streptothricin (250 μM) or AcCoA (650 μM) was present in the injection syringe. All samples were degassed for 20 min at 25°C before use. Injections (2.4 μl each) were made every 5 min into the chamber containing H₆-BsSatA under constant stirring at 350 rpm. ITC experiments were performed at 25°C. ITC data were analyzed using NanoAnalyze software (TA Instruments).