Reagents

All reagents and enzymes were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA), unless otherwise stated. Arsinothricin (AST) was purified from cultures of B. gladioli GSRB05, as described previously10. The concentration and purity of purified AST were determined by inductively coupled plasma mass spectrometry (ELAN DRC-e; Perkin–Elmer, Waltham, MA, USA) and high pressure liquid chromatography (series 2000, Perkin–Elmer) coupled to inductively coupled plasma mass spectrometry. AST is assumed to be the L-enantiomeric form based on the ArsN1 crystal structure with bound L-AST (vide infra). Commercial phosphinothricin (PPT) and methionine S-sulfoximine (MSO) are the D,L- and L-enantiomers, respectively. In the studies described below the concentration of D,L-PPT was divided by a factor of 2 to give the concentration of the L-enantiomer, the active form of the antibiotic13. Methylarsonous acid (MAs(III)) was prepared as described previously34.

Bacterial strains

Escherichia coli strains DH5α (Promega, Madison, WI, USA) and TOP10 (Invitrogen, Waltham, MA, USA) were used for gene cloning and protein expression, respectively. E. coli strain W311035 and the ars operon deleted derivative AW3110 (Δars)36, Pseudomonas putida KT2440 and the double ars operon deleted derivative strain (Δars1,2)21, Burkholderia gladioli GSRB059, Shinorhizobium meliloti Rm102137, Shewanella putrefaciens 200, Bacillus cereus UW8538, Bacillus megaterium (ATCC 14581), Corynebacterium glutamicum (ATCC 13032), Enterobacter cloacae (ATCC BAA-2341) and Mycobacterium bovis BCG (ATCC 19274) were used for in vivo resistance assay.

Cloning, expression and protein purification

For gene cloning and protein expression, E. coli cells were grown at 37 °C in lysogeny broth (LB) medium39 supplemented with 0.1 mg/ml ampicillin. For construction of a plasmid for expression of arsN1 from P. putida KT2440 (PparsN1) (accession number: AAN67541.1) in fusion with a six histidine tag at C-terminus, a 558-bp fragment excluding the stop codon was PCR-amplified from total genomic DNA of P. putida KT2440 by high fidelity PfuTurbo DNA polymerase (Agilent Technologies Inc., Santa Clara, CA, USA) using the forward primer 5′-CCAG CCATGG ATAGCGGAATCGATATTCG-3′ (NcoI site underlined) and reverse primer 5′-CCAG AAGCTT ACGAGGCACTGGGATTTGG-3′ (HindIII site underlined) and then ligated into pBAD-Myc/His-A as an NcoI/HindIII digest, generating the plasmid pBAD-PparsN1. The DNA sequence for pat, the gene encoding phosphinothricin N-acetyltransferase from Streptomyces viridochromogenes (Svpat) (accession number: AAU00088.1) with six histidine codons inserted at the 3’ end before the stop codon, was chemically synthesized by GenScript (NJ, USA) with 5′ NcoI and 3’ HindIII sites and cloned into the EcoRV site of pUC57-Kan (pUC57-Kan-Svpat). The synthetic Svpat gene was cloned as an NcoI/HindIII digest from pUC57-Kan-Svpat into pBAD-Myc/His-A, generating plasmid pBAD-Svpat. Cells of E. coli TOP10 bearing pBAD-PparsN1 or pBAD-Svpat were grown in LB medium with shaking at 37 °C. At an A 600nm of 0.5–0.6, L-arabinose was added as an inducer at a final concentration of 0.2% (w/v). After 5 h, the cells were harvested and stored at −80 °C until use. The frozen cells were thawed and washed once with and resuspended in buffer A (50 mM morpholinopropane-1-sulfonic acid, pH 7.5, containing 20 mM imidazole, 0.5 M NaCl and 20% (v/v) glycerol) (5 ml per gram of wet cells). The cells were lysed by a one-time passage through a French pressure cell at 20,000 psi and immediately mixed with 2.5 µl per g of wet cell of diisopropylfluorophosphate. The cell lysate was centrifuged at 40,000 rpm using a T865 rotor (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at 4 °C. The supernatant solution was applied onto a Ni-NTA column (QIAGEN Sciences, Hilden, Germany) at a flow rate of 1.0 ml/min and washed with 20 column volumes (100 ml) of buffer A. Bound protein was eluted with buffer A containing 0.2 M imidazole, and the purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis40. Protein concentrations were estimated by the Bradford assay by using bovine serum albumin as a standard. Fractions containing the protein were pooled and concentrated using a 10 kDa Amicon Ultra centrifugal filter (EMD Millipore, Billerica, MA, USA). The concentrated protein was rapidly frozen and stored at −80 °C until use.

Glutamine synthetase assays

The activity of glutamine synthetase from E. coli was measured using a coupled assay that determines formation of the product ADP to oxidization of NADH41. The 1 ml reaction mixture contained 34 mM imidazole, 9 mM ATP, 1 mM phosphoenolpyruvate, 60 mM magnesium chloride, 19 mM potassium chloride, 45 mM ammonium chloride, 0.25 mM NADH, 13 to 20 units of l-lactic dehydrogenase and 8–14 units of pyruvate kinase. The reaction was initiated by addition of glutamine synthetase at 0.2 nM, final concentration. The decrease in A 340nm was measured at 37 °C, and oxidation of NADH to NAD+ was quantified using an extinction coefficient 6230 M−1cm−1. The assays were performed with concentrations of l-glutamate from 2 to 100 mM. Inhibition constants (K i ) for AST and L-PPT were determined from the apparent K m of glutamine synthetase calculated with three different concentrations of inhibitor. Activities were corrected with the values from control assays without enzyme. Kinetic constants were calculated using Sigma Plot (Systat Software, Inc., Sun Jose, CA).

N-acetyltransferase assays

The enzymatic activity of purified PpArsN1 was measured from the rate of 5,5’-dithio-bis-2-nitrobenzoic acid reduction as described previously with minor modifications42. The reactions were carried out in 20 mM Tris-HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 0.33 mM 5,5′-dithio-bis-2-nitrobenzoic acid, 0.2 mM acetyl coenzyme A (AcCoA) with 50 µM AST, PPT or MSO at 37 °C. The reactions were initiated by addition of AcCoA, and the linear increase in A 412nm was measured over the first 2 min. The specific activity was determined using the molar extinction coefficient of 2-nitro-5-benzoatic acid (14,150 M−1 cm−1)42. Activities were corrected with the values from control assays without enzyme. The kinetics of PpArsN1 and SvPAT for PPT and AST were determined over a concentration range between 1 µM and 2 mM using 0.2 µM enzyme. Kinetic constants were calculated from a fit of the data to the Michaelis-Menten equation43 using SigmaPlot.

Bacterial-resistance assays

Middlebrook 7H9 broth (Difco Laboratories Inc., Detroit, MI, USA) supplemented with 5 g bovine serum albumin, 2 g dextrose, 0.85 g NaCl and 0.05% tween 80 (Fisher Scientific International Inc., Pittsburg, PA, USA) was used to culture M. bovis BCG. Mycobacterial cells were inoculated at a density of 1.0 × 105 CFU/ml and horizontally cultured in the presence or absence of the indicated concentrations of AST, L-PPT or L-MSO in an incubator humidified at 37 °C under 5% CO 2 for up to 4 weeks. Viable cells in each culture were determined by A 600nm . All other bacterial strains were grown in LB medium overnight, following which the cells were centrifuged, washed with and resuspended in M9 medium39 to an A 600nm of 0.04−0.06, with or without the indicated concentrations of As(III), MAs(III), AST, L-PPT or L-MSO. M9 medium was supplemented with 0.2% (w/v) citrate and 20 µg/ml uracil for P. putida strains, while M9 medium supplemented with 0.2% (w/v) glucose was used for the other bacterial strains. 0.1 mg/ml ampicillin and 0.2% (w/v) arabinose as inducer were added to cultures of E. coli, as required. Resistance was determined from the A 600nm after 24 h. E. coli and B. megaterium were grown at 37 °C. Other bacterial strains were cultured at 30 °C.

Cytotoxicity assays

Human acute monocytic leukemia THP-1 cells (ATCC TIB-202TM) were seeded in a 24-well plate (Nalge Nunc International, Rochester, NY, USA) with 300 µl of RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) fetal bovine serum and 0.05 mM 2-mercaptoethaol at a density of 1.0 × 105 cells/well and cultured in a 5% CO 2 humidified incubator at 37 °C. After 24 h, THP-1 cells were further cultured in the presence or absence of the indicated concentrations of AST or As(III) for another 24 h, following which viability of cells was determined by a 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay44. 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide was added to each well at a final concentration of 0.5 mM and the cultures were incubated for 3 h. The plate was then centrifuged at 400 × g, the cell pellets were lysed with 300 µl of dimethyl sulfoxide to dissolve 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide formazan. Cell viability was estimated from A 570nm.

ArsN distribution and phylogenetic analysis

The prevalence of arsN (arsN1 and arsN2, see Results) genes in ars operons was analyzed in representative organisms. GenBank accession numbers of the following bacterial genomes are given in parentheses. P. putida KT2440 (AE015451), Bacillus sp. GZT (LVVJ00000000), Meiothermus chliarophilus DSM 9957 (AUQW00000000), Acidovorax sp. CF316 (AKJX00000000), Porphyrobacter mercurialis (JTDN00000000), Pseudomonas syringae pv. syringae B728a (CP000075), Inquilinus limosus DSM 16000 (AUHM00000000), Sphingopyxis sp. KK2 (LYVN00000000), Sphingomonas yabuuchiae (LDTF00000000), Pelomonas sp. KK5 (LYVQ00000000), Burkholderia lata (CP000150), Rhodobacter sphaeroides ATCC17025 (CP000661), Rubrobacter xylaniophilus DSM 9941 (CP000386), Mumia flava (JTDJ00000000), Roseiflexus castenholzii DSM 13941 (CP000804), Ralstonia pickettii 12 J (AAWK00000000), Rhizobium leguminosarum bv. viciae 3841 (AM236080), Burkholderia phytofirmans PsJN (AAUH00000000), Spirosoma panaciterrae DSM 21099 (ARFA00000000), Oxalobacteraceae bacterium AB_14 (ARMC00000000), Geobacillus kaustophilus HTA426 (BA000043), Paenibacillus stellifer DSM 14472 (NZ_CP009286), Thermoflavimicrobium dichotomicum DSM 44778 (NZ_FORR01000002), Deinococcus aquatilis DSM 23025 (NZ_KB899704), Meiothermus terrae DSM 26712 (NZ_QXDL01000023). Multiple alignment of the sequences of putative N-acetyltransferase orthologs was performed using T-Coffee45 and BoxShade46. N-acetyltransferase sequences distributed in ars operons20,21 were defined as ArsN [P. putida KT2440 (WP_010952945), Bacillus sp. GZT (WP_062922891), M. chliarophilus DSM 9957 (WP_027893731, WP_027893733), Acidovorax sp. CF316 (WP_007857208), P. mercurialis (WP_039093634), P. syringae pv. syringae B728a (YP_234588), I. limosus DSM 16000 (WP_026871525), Sphingopyxis sp. KK2 (WP_077145629), S. yabuuchiae (WP_058746515, WP_058746517), Pelomonas sp. KK5 (WP_077035561), B. lata (WP_011349260), R. sphaeroides ATCC 17025 (WP_011908437), R. xylanophilus DSM 9941 (WP_011565797), M. flava (KHL15495), R. castenholzii DSM 13941 (WP_012120818), R. pickettii 12 J (WP_012429982), R. leguminosarum bv. viciae 3841 (WP_011652407), B. phytofirmans PsJN (WP_012431288), S. panaciterrae DSM 21099 (WP_020601039) and O. bacterium AB_14 (WP_020703167), G. kaustophilus HTA426 (BAD74878), M. terrae DSM 26712 (WP_119314079), P. stellifer DSM 14472 (WP_038700913), T. dichotomicum DSM 44778 (WP_093227883) and D. aquatilis DSM 23025 (WP_019009361)]. N-acetyltransferase sequences distributed in phosphinothricin tripeptide biosynthesis gene clusters27 were defined as phosphinothricin N-acetyltransferase (PAT) [Streptomyces hygroscopicus (P16426), Streptomyces viridochromogenes (WP_003988626), Kitasatospora phosalacinea NRRL B-16230 (KP185121) and Actinobacteria bacterium OK074 (WP_082414639)]. N-acetyltransferases with higher selectivity for PPT compared to MSO47,48,49 are also defined as PAT [Streptomyces coelicolor A3(2) (CAB90987), Rhodococcus sp. YM12 (JQ398613) and Nocardia sp. AB2253 (BAG06876)]. N-acetyltransferases with higher selectivity on MSO compared to PPT21,47,50,51 are defined as methionine sulfoximine N-acetyltransferase (MAT) [E. coli K-12 (AAC74530), Salmonella enterica Typhimurium str. LT2 (NP_460549), P. putida KT2440 (WP_010955452) and Pseudomonas aeruginosa PAO1 (AAG08251), Acinetobacter sp. ADP1 (Q6FBS8)]. N-acetyltransferases that have similar activity on both PPT and MSO47 are also included [Geobacillus kaustophilus HTA426 (BAD77205), Bacillus subtilis RO-NN-1 (AEP92705), Paraburkholderia xenovorans LB400 (ABE30708, ABE34181), Staphylococcus aureus USA300_FPR3757 (ABD22256) and Deinococcus radiodurans R1 (AAF10750)]. GenBank accession numbers of N-acetyltransferase orthologs are given in parentheses. Phylogenetic analysis was performed to infer the evolutionary relationship among the sequences of ArsN1, PAT and/or MAT from various organisms. The phylogenetic tree was constructed using the Neighbor-Joining method using MEGA X52. The statistical significance of the branch pattern was estimated from a 1000 bootstrap53.

Crystallization and structure determination

Initial crystallization screening was performed as described previously54 by the sitting-drop vapor-diffusion method55 using a variety of crystal screens from Hampton Research (Aliso Viejo, CA, USA), Emerald BioSystems, Inc. (Bainbridge Island, WA, USA) and Jena Bioscience GmbH (Jena, Germany) in 96-well plates (Corning Inc., Corning, NY, USA) at 293 K. Crystalline precipitates were obtained at 0.2 M sodium acetate, 0.1 M Tris-HCl, pH 8.5, and 30% (w/v) PEG 4000. Diffraction quality crystals were grown using the vapor diffusion hanging drop method in 24-well Linbro plates. The reservoir solution (0.3 ml) consisted of 0.2 M sodium acetate, 0.1 M Tris-HCl and 20% (w/v) PEG 6000, and the hanging drop contained 2 µl of 20 mg/ml of purified PpArsN1, 2 µl reservoir solution and 1 µl of 0.1 M ATP. Rod-shaped crystals, with approximate dimensions of 0.1 × 0.05 × 0.05 mm, were obtained within a few weeks. The PpArsN1-AST complex was prepared by adding 0.5 ml of 4.0 mM AST to 0.5 ml of 1 mM protein. PpArsN1-AST crystals were grown using vapor diffusion hanging drop method. The hanging drop contained 2 µl of PpArsN1-AST complex and 2 µl of reservoir solution. The reservoir contained 1.5 M sodium formate and 0.1 M sodium acetate with pH 4.5. Thin plate-like crystals were obtained within a week. The PpArsN1-PPT complex was prepared by adding 0.5 ml of 25 mM L-PPT to 0.5 ml of 1 mM protein. The PpArsN1-PPT crystals were also grown using the same method and crystallization condition as used for the PpArsN1-AST crystals. The crystals were harvested from the hanging drop using a cryoLoop, flash-frozen in liquid nitrogen at 100 K and stored in liquid nitrogen. Ethylene glycol (20%, v/v) was used as cryoprotectant. X-ray data were collected on beamline 22ID at the Advanced Photon Source), Argonne National Laboratory, using a MAR300HS detector. The crystal-to-detector distance was 180 mm, and 180 images for PpArsN1 crystal, 240 and 360 images for PpArsN1-AST and PpArsN1-PPT crystals, respectively, were collected with 1° oscillations. The PpArsN1 diffraction data were indexed and scaled using KYLIN56 and PpArsN1-AST and PpArsN1-PPT data were indexed and scaled using HKL200057. The data processing statistics are shown in Table 2. The PpArsN1 crystal diffracted to 2.16 Å resolution. The crystal belongs to space group P4 3 2 1 2 with cell dimensions a = b = 67.02 Å, c = 206.74 Å. The Matthews coefficient of 2.48 indicates that there are two molecules in the asymmetric unit with 50.5% solvent. An initial homology model was constructed by molecular replacement with an acetyltransferase from P. aeruginosa PA01 (PDB ID: 1YVO as a template with 32.3 % identity) using SWISS-MODEL58. Molecular replacement was done using PHASER59 in the CCP4 suite60. The initial R and R free were 35.0 and 40.0%, respectively. The structure was refined using PHENIX61. The C-terminal extended residues were fitted in electron density using COOT62. Water molecules were added at appropriate positions and refined. The final R and R free are 23.7% and 26.6%, respectively. The PpArsN1-AST crystal diffracted to 2.19 Å resolution and indexed with C121 space group with cell dimensions a = 185.27, b = 141.74, c = 54.55 Å and β = 90.6°. The Matthews coefficient of 2.54 indicates that there are six molecules in the asymmetric unit with 51.6% solvent. The PpArsN1-apo structure was used as a model for molecular replacement. There are positive electron densities at the 9.0 and 16.0 σ level near Arg77 in molecule A and B, respectively (Supplementary Figure 9). The density was fitted with the L-enantiomer of AST, and the anomalous difference map confirmed the presence of arsenic. The PpArsN1-PPT crystal diffracted to 2.66 Å resolution and indexed with P12 1 1 space group with cell dimensions a = 53.84, b = 142.69, c = 178.31 Å and β = 89.9°. The Matthews coefficient of 2.45 indicates that there are twelve molecules in the asymmetric unit with 49.8% solvent. The PpArsN1-apo structure was used as model for molecular replacement. There are positive electron densities between 6.5 and 9.0 σ level near Arg77 in molecule A–D, G–J (Supplementary Figure 10). The density was fitted with two L-PPT molecules. The structure were refined using REFMAC563 in the CCP4 suite60. The simulated annealing refinement was done using PHENIX. The structure factor and coordinates were deposited to the Worldwide Protein Data Bank (wwPDB, accession IDs: 5JTF (PpArsN1), 5WPH (PpArsN1-AST) and 6M7G (PpArsN1-PPT)). The molecules were drawn with PyMol (Version 1.8 Schrödinger, LLC). Docking was performed using AutoDockTools and AutoDock464.

Table 2 Data collection and refinement statistics Full size table

Statistics

Assays of glutamine synthetase, N-acetyltransferase, bacterial resistance and cytotoxicity were repeated at least three times. The data are presented as the mean ± standard error (SE). No other statistical tests were performed.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.