Antibiotics and reagents

All reagents were purchased from Sigma-Aldrich (Oakville, Ontario, Canada) unless otherwise specified. Acetyl-coenzyme A (CoA) was purchased from BioShop (Burlington, Ontario, Canada). Organic solvents were purchased from Fisher Scientific (Ottawa, Ontario, Canada). Telithromycin was purified from the pharmaceutical formulation Ketek (400 mg, Sanofi-Aventis US). Briefly, pills were crushed in a mortar and pestle, dissolved in acetonitrile at 40 °C, passed through an Amicon Ultra-15 10 kDa centrifugal filter unit and lyophilized. Linopristin and flopristin were gifts from AstraZeneca.

Growth conditions and genomic DNA isolation

Paenibacillus sp. LC231 was isolated from Lechuguilla Cave, as described13. To identify the potential for contamination by surface bacterial isolates, culture and preparation controls were used throughout sampling. Culture controls consistent of diluent and culture media mock-inoculated in the cave with sterile swabs, while preparation controls included all prepared media. All controls were incubated in the laboratory for 30 days under cave-relevant conditions (20 °C in the dark). None of the controls demonstrated any microbial growth, confirming that all observed colonies represented indigenous cave isolates. P. lautus ATCC 43898 was obtained from Cedarlane (Burlington, Canada). Paenibacillus strains were streaked from glycerol stocks (−80 °C) onto Tryptic Soy Agar and incubated at 30 °C for 2 days and cultures were re-streaked a second time before use. Several colonies with different growth phenotypes (that is, stationary and motile colonies) were always used for the experiments to ensure reproducibility. Paenibacillus vortex is a closely related strain that is also motile. Previous work in this strain has demonstrated that the stationary and motile colonies can have different antibiotic resistance phenotypes23. We found that using multiple colonies increased the reproducibility of our experiments. Liquid cultures were incubated with shaking (250 r.p.m.) at 30 °C for 2 days unless otherwise specified. The cell pellet from 3 ml of Paenibacillus sp. LC231 cultured in tryptic soy broth (TSB) was used for genomic DNA isolation using a PureLink genomic DNA mini kit (Invitrogen). E. coli TOP10 (Invitrogen) was cultured in LB-Lennox and ampicillin (100 μg ml−1) or kanamycin (50 μg ml−1) were added to cultures harbouring plasmids. S. aureus RN4220 (gift from Dr Eric Brown, McMaster University) was cultured on LB agar, and Staphylococcus saprophyticus ATCC 15305 and a strain of K. rhizophila isolated from the soil by our lab (16s rRNA is 99% identical to K. rhizophila ATCC 9341) were cultured on Tryptic Soy Agar. E. coli strains were cultured in LB-Lennox (10 g typtone, 5 g yeast extract and 5 g NaCl).

Genome sequencing and resistance prediction using the RGI

The Paenibacillus sp. LC231 genome was sequenced using an Illumina MiSeq with the Reagent Kit v2 and paired-end 250 bp reads. Spades v3.5.0 was used to assembly the genome. The Resistance Gene Identifier (RGI) v3 (beta, accessed on 21 August 2015) was used to annotate resistance genes based on the curated CARD15.

Antimicrobial susceptibility and inactivation assays

Antimicrobial susceptibility experiments followed Clinical and Laboratory Standards Institute (CLSI) guidelines for determining the MIC of compounds using the broth microdilution method24 with the following modifications. For Paenibacillus strains, the saline suspension was made from several colonies with different growth phenotypes to ensure reproducibility. Ninety-six-well plates were incubated without shaking at 30 °C for 2 days (for Paenibacillus and K. rhizophila) or overnight at 37 °C (S. aureus and E. coli). Each MIC was performed in duplicate. Inactivation experiments for Paenibacillus sp. LC231 were set-up in the following way except for kasugamycin and capreomycin (see below). Antibiotics were added at ¼ MIC to 3 ml of Mueller Hinton Broth (MHB) and inoculated as described above but with shaking. Cells were removed by centrifugation at 21,000g for 5 min and the supernatant was assayed for antibiotic activity using a Kirby–Bauer disc diffusion assay and K. rhizophila or S. saprophyticus as the indicator organism. Inactivation was defined as a significant decrease in the zone-of-inhibition when compared with the sterile control.

Capreomycin and kasugamycin inactivation experiments were performed using Paenibacillus sp. LC231 lysate. A 250 ml culture in TSB was centrifuged at 10,000g and the cell pellet was washed with saline. The pellet was resuspended in 5 ml of 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl 2 and 1 mM β-mercaptoethanol, and cells were lysed at 30,000 psi using a One Shot cell disrupter (Constant Systems, Ltd.). The lysate was clarified by centrifuging at 18,000g for 20 min. Inactivation of capreomycin and kasugamycin was evaluated in a 50 μl reaction and the following conditions: 25 μl of clarified lysate in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA and 0.5 mg ml−1 of capreomycin or kasugamycin. The reactions were incubated at room temperature overnight and an equal volume of cold methanol was added. Samples were stored at −20 °C overnight and then centrifuged at 21,000g. Control experiments used 25 μl of clarified lysate that was boiled for 10 min. Antibiotic modification was evaluated using LC–MS. Reactions were analysed by injecting 10 μl onto an Agilent 1100 Series LC system and a QTRAP LC/MS/MS System (ABSciex). The reverse-phase HPLC conditions are as follows: isocratic 5% solvent B (0.05% formic acid in acetonitrile), 95% solvent A (0.05% formic acid in water) over 1 min, followed by a linear gradient to 97% B over 7 min at a flow rate of 1 ml min−1 and C18 column (Sunfire, 5 μm, 4.6 × 50 mm).

Paenibacillus sp. LC231 library construction

Genomic DNA (20 μg) was fragmented to 5 kb with a Couvaris S220 focused-ultrasonicator using red Minitubes (Covaris). Sheared DNA was end repaired with Fast DNA End Repair Kit (Thermo Scientific) according to the manufacturer’s protocol. The End-Repair reaction (200 μl) contained 50 μl sheared DNA, 20 μl buffer and 10 μl enzyme mix. The repaired DNA was run on a 1% low-melting point agarose gel (1 × TAE) and fragments between 3 and 8 kb were excised using the GeneJET gel extraction kit (Thermo Scientific). Fragmented DNA was further purified using a GeneJET PCR purification kit (Thermo Scientific). Fragments were ligated into the SmaI restriction site of pUC19 in a 30 μl reaction (1.5 μl T4 DNA ligase (Thermo Scientific), 6 μl Fast Ligation buffer (Thermo Scientific), 120 ng SmaI digested pUC19 and 810 ng fragmented DNA) and incubated at room temperature for 2 h. E. coli ElectroMax DH10B electrocompetent cells (200 μl, Invitrogen) were transformed by electroporation with 5 μl of the ligation reaction and recovered in 9 ml of SOC media for 2 h at 37 °C and then plated evenly across 15 LB agar plates with ampicillin and incubated at 37 °C overnight. A measure of 5 ml of LB was added to each agar plate to facilitate scraping of E. coli clones. Resuspended cells from all plates were combined, washed in LB and resuspended in 25 ml LB with 15% glycerol and stored in 1 ml aliquots at −80 °C. Average fragment size was estimated by purifying plasmids from 12 clones and PCR amplifying the inserts using Phusion DNA polymerase (Thermo Scientific) with M13 forward and reverse sequencing primers.

Selection of antibiotic resistant clones

The Paenibacillus sp. LC231 genomic library in E. coli was plated at a density of 6–7 × the number of unique clones obtained in the transformation experiment. A measure of 3 μl of the library glycerol stock was diluted in 3 ml of warm LB and incubated with shaking for 1 h at 37 °C. Then, 200 μl was spread on pre-warmed LB agar plates with binary combinations of 100 μg ml−1 ampicillin and one of the following antibiotics: 20 μg ml−1 tetracycline, 50 μg ml−1 kanamycin, 400 μg ml−1 capreomycin, 400 μg ml−1 kasugamycin, 500 μg ml−1 tiamulin, 200 μg ml−1 clindamycin and 100 μg ml−1 mupirocin. Plates were incubated at 37 °C overnight with the exception of clindamycin, which was incubated an additional day at room temperature. Resistant clones were confirmed by replica plating onto media with 1–4 × the antibiotic concentration. Unique clones were identified using colony-PCR with M13 forward and reverse sequencing primers. Pure plasmids were submitted for Sanger sequencing (MOBIX, McMaster University, Canada). The results of Sanger sequencing were mapped to the assembled genome visualized in Geneious. The complete nucleic acid sequence of the insert was inferred from the genomic region between the mapped Sanger reads. Enzyme function of potential resistance enzymes (for example, acetyltransferase and hydrolase) was predicted by matching the protein sequence to a Pfam25.

Identification of BahA

E. coli is naturally insensitive to bacitracin. The library glycerol stock described above was diluted 2 × 10−6 in LB and 200 μl was spread on 35 LB agar plates containing ampicillin and incubated overnight at 37 °C to produce ∼300-well separated colonies. Each plate was replica plated onto two additional plates and incubated overnight at 37 °C. One plate, to be used to pick bacitracin-inactivating clones, contained ampicillin and was stored at 4 °C. The other plate lacked ampicillin and was overlaid with 5 ml of MHB with 0.75% agar and 4 μg ml−1 zinc bacitracin. These plates were further incubated at 37 °C overnight to permit bacitracin inactivation by a clone expressing a bacitracin-modifying enzyme. An overnight culture of K. rhizophila was diluted one in three in sterile saline and spread on each plate using a cotton swab, which was then incubated at 30 °C for an additional two days. The bacitracin concentration used in this experiment is 2 × the MIC of K. rhizophila under these conditions and completely inhibits colony formation. E. coli clones with a halo of yellow K. rhizophila growth were identified as expressing a bacitracin-inactivating enzyme. To confirm this result, the corresponding colonies from the replica plate were used to inoculate 3 ml of LB with 200 μg ml−1 zinc bacitracin. BahA was identified by Sanger sequencing.

Construction of a Paenibacillus species tree

A Paenibacillus species tree was generated using 10 housekeeping genes; atpD, dnaA, gyrB, pgi, pyrH, recA, rpoB, sucC, topA and trpB26. Sequenced Paenibacillus strains with full sequences for all 10 housekeeping genes were used in phylogenetic analysis, except Paenibacillus vortex, which did not have an intact topA sequence (Supplementary Table 11)27. Bacillus megaterium DSM319 (CP001982.1) and Brevibacillus brevis NBRC 100599 (AP008955.1) were used as an outgroup. Each housekeeping gene was aligned with MAFFT (L-INS-i)28, trimmed with TrimAl v. 1.4 rev15 using the automated1 setting29, and concatenated. A maximum-likelihood tree was generated using RAxML with rapid Bootstrap analysis on 1,000 replicates. 'X's were used in place of the missing Paenibacillus topA sequence. All genome sequences were accessed on 10 July 2015.

Cloning of resistance genes and susceptibility testing

Cloning is summarized in Supplementary Table 12. TaeA and TetAB(48) are ABC-transporters composed of one and two polypeptides, respectively. Primers were designed to amplify 500 bp upstream of tetAB(48) so to clone both genes with their native promoter in pUC19. Similarly, taeA was cloned into pUC19 with the 500 bp upstream region. E. coli TOP10 expressing either TetAB(48) or TaeA were used in MIC experiments. The C-terminal domain of bahA (corresponding to residues 200–756) was first cloned into pET11a, then PCR-amplified with the upstream ribosome-binding site and XbaI-restriction site and cloned into pET21a, which includes an in frame C-terminal His 6 -tag. The pET11a-bahA construct was transformed into E. coli BL21(DE3) for MIC experiments. In bacitracin inactivation experiments, 3 ml of LB containing 200 μg ml−1 bacitracin was inoculated with either E. coli BL21(DE3) pET11a or E. coli BL21(DE3) pET11a-bahA and cultured with shaking overnight. Inactivation was assayed using a Kirby–Bauer assay and using LC–electrospray ionization (ESI)–MS as described above. The pET21a constructs of aac(2′)-IIb and aac(6′)-34 were used for MIC experiments. All pET vectors were transformed into E. coli BL21(DE3) for MIC experiments.

Pan-Paenibacillus resistance enzyme analysis

Orthologues of ten resistance enzymes from Paenibacillus sp. LC231 (RphB, VatI, AAC(2′)-IIb, AAC(6′)-34, MphI, CatU, VgbC, LlmA, CpaA and BahA) were identified in a subset of Paenibacillus genomes using an extended BLASTp method. Briefly, each protein sequence (for example, RphB and VatI) was queried against GenBank limited to txid44249 (Paenibacillus) with an e-value cutoff of 1 × 10−10. Enzymes that were at least 50% identical over at least 80% of the seed sequence were in turn queried against GenBank to identify more diverse Paenibacillus orthologues using the same BLAST parameters. The pair-wise sequence identity of each enzyme with its orthologue in Paenibacillus sp. LC231 was computed with Clustal Omega30 per cent identity matrix and plotted as a colour gradient from 45% (the most diverse orthologue) in black to 100% in red. For genomic context analysis, sequence and annotations for 5 kb upstream and downstream of each gene were extracted from GenBank files using RefSeq annotations. Arrows representing genes were made in Geneious. Genes from Paenibacillus sp. LC231 were manually annotated using BLASTx.

Purification of antibiotic inactivating enzymes

Antibiotic-inactivating enzymes were overexpressed in E. coli BL21(DE3) for protein overexpression. A 3 ml overnight culture was used to inoculate 1 l of LB and incubated with shaking at 37 °C with either kanamycin (pET28a) or ampicillin (pET11a, pET21a or pET22b) until an OD 600 of 0.6–0.8, at which point the cultures were placed in an ice bath for 20 min and protein expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside at 16 °C overnight. Cells were collected at 5,000g for 20 min and washed in cold saline. The cell pellet was resuspended in 20 ml of 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 5 mM imidazole (buffer A) and lysed using a One Shot cell disrupter (Constant Systems, Ltd.) at 20,000 psi. A measure of 5 mg of bovine pancreas DNase, 2.5 mg of bovine pancreas RNase and an additional 15 ml of buffer were added and centrifuged at 40,000g for 45 min. Overexpressed proteins were purified using ion-metal affinity chromatography (1 ml Ni2+-nitrilotriacetic acid column, Qiagen) equilibrated with buffer A. A linear gradient was used to elute protein over 20 column volumes starting at 94:6 buffer A: buffer B to 100% buffer B (50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 250 mM imidazole) on an ÄKTA purifier (GE Scientific). Fractions containing pure protein were identified using SDS–polyacrylamide gel electrophoresis, pooled and desalted with 50 mM HEPES pH 7.5 using a PD-10 gel filtration column (GE), except for CatU and VatI where the buffer contained 150 mM NaCl. In addition, 0.1 mM β-mercaptoethanol was added to purified CatU. Pure enzyme stocks were stored at 4 °C.

MS of antibiotics inactivated by purified enzymes

The predicted catalytic functions of CpaA, VatI, MphI, CatU, VgbC, RphB, AAC(2′)-IIb and AAC(6′)-34 were confirmed with LC–MS analysis of enzyme reactions. Each 250 μl reaction consisted of 1 mg ml−1 antibiotic, 60–90 μg ml−1 of enzyme and 1 × reaction buffer (see below). Specific enzyme/substrate combinations were as follows; CpaA/capreomycin, VatI/flopristin, MphI/telithromycin, CatU/chloramphenicol, VgbC/linopristin, RphB/rifampin, AAC(2′)-IIb/kasugamycin and AAC(6′)-34/sisomicin. The reaction for CpaA, VatI, CatU and AAC(2′)-IIb contained 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA and 1 mM acetyl-CoA. The MphI reaction contained 50 mM HEPES pH 7.5, 40 mM KCl, 10 mM MgCl 2 and 1 mM GTP. The reaction for VgbC contained 50 mM HEPES pH 7.5 and 1 mM MgCl 2 . The reaction for RphB contained 50 mM HEPES pH 7.5, 40 mM NH 4 Cl, 5 mM MgCl 2 and 2.5 mM ATP. The AAC(6′)-34 reaction contained 25 mM MES pH 6.0, 1 mM EDTA and 1 mM acetyl-CoA. Reactions were incubated at room temperature overnight. An equal volume of cold methanol was added, stored at −20 °C overnight and centrifuged at 21,000g for 10 min. A volume of 10–20 μl of each reaction was injected onto an Agilent 1100 Series LC system and a QTRAP LC/MS/MS System using the HPLC conditions described in online methods.

Structural characterization of inactivated bacitracin

A measure of 0.5 ml of a 3 ml overnight Paenibacillus sp. LC231 culture in MHB was used to inoculate 50 ml of MHB. The culture was centrifuged at 4,000g for 40 min at room temperature, washed with 10 ml of sterile saline and resuspended in 1.5 ml MHB. A volume of 750 μl of this suspension was boiled for 10 min as a negative control. Bacitracin A (2.5 mg ml−1) was added to the suspension and incubated overnight with shaking at 30 °C. Cells were removed by centrifuging at 21,000g. An equal volume of methanol was added, stored at −20 °C overnight and centrifuged at 21,000g for 10 min. LC–ESI–MSn experiments were performed on a ThermoFisher LTQ-XL-Orbitrap Hybrid mass spectrometer (ThermoFisher, Bremen, Germany) in positive-ion mode. A volume of 20 μl of the reaction or bacitracin A (1 mg ml−1) were injected onto an Agilent 1290 Infinity UPLC system using the following chromatography conditions: isocratic 5% solvent B (0.05% formic acid in acetonitrile), 95% solvent A (0.05% formic acid in water) over 1 min, followed by a linear gradient to 100% B over 4.5 min at a flow rate of 0.4 ml min−1 and C18 column (info needed). The conditions for ESI–MSn experiments are as follows: sheath gas flow rate at 40, auxiliary gas flow rate at 10, ion spray voltage at 4.2 kV, capillary temperature at 340 °C, capillary voltage at 29 V, tube lens voltage at 120 V and normalized collision energy at 27%. MS1 was set to fourier transform (FT) full scan between 500–1,700 m/z with a resolution set at 60,000 followed by MS2 and MS3 of the most intense ions from MS1 and MS2 stages, respectively. Ion fragments in MS3 were compared with previous reports (Supplementary Table 5)31.

CpaA characterization

CpaA substrate specificity was examined by monitoring CoA liberation with 4,4′-dithiodipyridine (DTDP) at 324 nm. Reactions were performed at 25 °C in a 96-well plates with a final volume of 250 μM and contained 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1.7 mM DTDP, 250 μM acetyl-CoA and 5.6 μg ml−1 CpaA. Reactions were initiated with the addition of 100 μM capreomycin, kanamycin or viomycin and monitored for 1,000 s. The site of acetylation was determined using NMR. A large-scale reaction consisted of 25 mM ammonium bicarbonate pH 7.8, 12 mg capreomycin (mixture of IA and IB), 15 mM acetyl-CoA, and 14 μg CpaA in a final volume of 1 ml and was incubated at 25 °C overnight. An additional 10 mM acetyl-CoA and 28 μg CpaA was added, and the reaction was incubated for 8 h. The solution was lyophilized, dissolved in 0.5 ml of 5 mM ammonium bicarbonate and the pH was adjusted to 9.0 using ammonium hydroxide. The sample was purified by anion-exchange chromatography (1 ml Q-sepharose HiTrap XL) and the compound was eluted in 5 mM ammonium bicarbonate pH 9.0. The eluate was lyophilized and the compound was further purified using normal-phase flash chromatography (12 g RediSep Rf silica, Teledyne) and eluted with butanol:acetic acid:water using a linear gradient from 3:1:1 to 3:3:4 to yield 10 mg acetyl-capreomycin. The compound was dissolved in 500 μl D 2 O and 1 μl glacial acetic acid for one-dimensional and two-dimensional NMR experiments.

The molecular formula of 1-N-acetyl-capreomycin IA was determined as C 27 H 46 N 14 O 9 according to its positive HRESIMS at m/z 711.3633 [M+H]+. Careful comparison of the 1H-NMR data of 1-N-acetyl-capreomycin IA with those of capreomycin IA showed that H-1 in 1-N-acetyl-capreomycin IA shifted to downfield 0.23 ppm, H-2a and H-2b shifted to upfield 0.20 and 0.24 p.p.m., respectively (Supplementary Fig. 3; Supplementary Tables. 7 and 8). In the 13C-NMR spectrum, C-16 shifted to downfield 3.9 p.p.m., indicating that a acetyl group connected to N-1. This connection was further confirmed by key heteronuclear multiple-bond correlation spectroscopy correlations from H-1 (at δ H 4.60 p.p.m.) to CH 3 C=O (at δ C 174.0 p.p.m.).

MphI characterization

MphI substrate specificity was examined using a coupled assay32. Reactions were performed in a 96-well plate with a final volume of 250 μl and contained 50 mM HEPES pH 7.5, 40 mM KCl, 10 mM MgCl 2 , 0.3 mM NADH, 3.5 mM PEP, 4.8 U PK/LDH, 1 mM GTP and 800 μM macrolide (erythromycin, clarithromycin, azithromycin, tylosin, telithromycin, spiramycin and roxithromycin). The assay plate was incubated at 37 °C for 5 min and reactions were initiated by the addition of GTP. Acid hydrolysis was used to remove the C3-cladinose from clarithromycin. Clarithromycin (19.7 mg, 26.3 mmol) was added in several portions over 5 min to a mixture of 500 μl water and 50 μl concentrated HCl. The reaction proceeded at room temperature, with stirring, for 2 h. The crude material was then purified using reverse-phase flash chromatography (C18, 5.5 g RediSep Rf Gold column, Teledyne) in a linear gradient of 100% water to 60:40 water:acetonitrile over 10 min. The fractions containing pure compound, as determined by LC–MS, were pooled and lyophilized. The compound structure was confirmed to be descladinose clarithromycin using one-dimensional and two-dimensional NMR experiments (Supplementary Figs 4 and 5; Supplementary Table 9). The yield was 58.4%. Descladinose clarithromycin was used in an MphI enzyme assay without PK:LDH in the following reaction; 0.5 mg ml−1 of descladinose clarithromycin, 22.5 μg MphI and 1 mM GTP in a final volume of 100 μl. A 50 μl aliquot was removed immediately after mixing and the reaction was stopped with an equal volume of cold methanol. The remainder of the reaction was incubated at 37 °C for 16 h. A negative control with clarithromycin was used for comparison. Each sample was analysed by LC–MS.

Kinetic characterization of antibiotic inactivating enzymes

Enzyme kinetics experiments were performed in triplicate except where noted. All reactions were initiated by adding enzyme except for MphI, which was initiated by adding GTP. All enzyme reactions were performed in 96-well Nunc plates (Thermo Scientific) in a Spectramax Plus384 (Molecular Devices) microtitre plate reader. GraphPad Prism was used for data analysis.

Steady-state kinetics of RphB was performed at 25 °C by monitoring inorganic phosphate production using the EnzChek Phosphate Assay Kit (Molecular Probes) in 50 mM HEPES pH 7.5, 40 mM NH 4 Cl, 5 mM MgCl 2 with a final volume of 100 μl16. The K m of ATP was determined as follows; 25 μM rifampin, 7.81–500 μM ATP and 68.5 nM RphB. The K m of rifampin was determined as follows; 1 mM ATP, 0.225–25 μM rifampin and 68.5 nM RphB. Enzyme reactions were performed in duplicate.

Steady-state kinetics of MphI was performed at 25 °C and in 50 mM HEPES pH 7.5, 40 mM KCl, 10 mM MgCl 2 in a volume of 250 μl. The K m of GTP was determined as follows: 400 μM tylosin, 3.12–2,000 μM GTP and 430 nM MphI. The K m of macrolides were determined as follows: 200 μM GTP, 2.34–800 μM macrolide and 430 nM MphI.

Steady-state kinetics of VgbC was performed by monitoring the decrease in absorbance associated with the linearization of streptogramin B antibiotics33. Enzyme reactions were performed at 25 °C and in 50 mM HEPES pH 7.5 and 1 mM MgCl 2 in a volume of 250 μl. Linopristin was used as the substrate and the decrease in absorbance was monitored at 305 nm (ɛ=6,220 M−1cm−1). For the K m of linopristin: 2.34–200 μM linopristin and 29.6 nM VgbC.

Steady-state kinetics of CatU was performed by monitoring CoA liberation using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) at 25 °C (ref. 34). Reactions were performed in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA and 1 mM DTNB in a final volume of 200 μl. The K m of acetyl-CoA was performed as follow: 400 μM chloramphenicol, 31.3–2,000 μM acetyl-CoA and 62.5 nM CatU. The K m of chloramphenicol was performed as follows: 500 μM acetyl-CoA, 4.69–500 μM chloramphenicol and 62.5 nM CatU. Acetyl-CoA was not saturating under these conditions.

Steady-state kinetics of VatI were performed in a volume of 250 μl at 25 °C and in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA35. The K m of acetyl-CoA was performed as follows: 100 μM flopristin, 7.81–1,000 μM and 13.8 nM VatI. The K m of flopristin was performed as follows: 250 μM acetyl-CoA, 3.91–500 μM flopristin and 13.8 nM VatI.

Steady-state kinetics of AAC(6′)-34 was performed by monitoring CoA liberation using DTDP in a 250 μl reaction at 25 °C containing 25 mM MES pH 6.0, 1 mM EDTA36. The K m of acetyl-CoA was performed as follows: 35 μM sisomicin, 18.8–1,000 μM acetyl-CoA and 120 nM AAC(6′)-34. The K m of sisomicin was determined as follows: 500 μM acetyl-CoA, 1.40–250 μM sisomicin and 120 nM AAC(6′)-34.

Steady-state kinetics of AAC(2′)-IIb were performed similar to AAC(6′)-34, but in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA in a volume of 250 μl (ref. 21). The K m of acetyl-CoA was performed as follows: 250 μM kasugamycin, 1.56–1,000 μM acetyl-CoA and 21.2 nM AAC(2′)-IIb. The K m of kasugamycin was performed as follows: 125 μM acetyl-CoA, 7.81–1,000 μM and 21.2 nM AAC(2′)-IIb. A similar assay was employed to examine the substrate specificity of AAC(2′)-IIb; 250 μM acetyl-CoA, 21.2 nM AAC(2′)-IIb and 50 μM of either kasugamycin, apramycin, ribostamycin, fortimicin A, gentamicin, paromomycin, lividomycin, amikacin or neomycin.

CpaA steady-state kinetics was performed at room temperature in 250 μl and in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA. The K m of acetyl-CoA was performed as follows: 5 μM capreomycin, 5.63–500 μM acetyl-CoA and 37.5 nM CpaA. The K m of capreomycin was performed as follows: 250 μM acetyl-CoA, 0.19–10 μM capreomycin and 37.5 nM CpaA.

Data availability

Nucleotide sequences for the Paenibacillus sp. LC231 genome have been deposited in the GenBank WGS database with the accession code JFOM00000000. The resistance determinants studied here were deposited in the GenBank Nucleotide database with accession codes KX531043 to KX531056. In addition, each resistance determinant was deposited in CARD (https://card.mcmaster.ca/). The authors declare that all other data supporting the findings of the study are included in this published article and its Supplementary Information files, or are available from the corresponding author on request.