Plasmids, bacterial Strains, media, and growth conditions
Plasmids used in this study are listed in Table S1. Staphylococcus xylosus ATCC 700404 was purchased from the American Type Culture Collection (ATCC) and preserved in our lab. All tylosin-resistant S. xylosus were also kept in our lab. All mutants of S. xylosus were constructed in this study. The above strains (Table S2) are cultured in Tryptic Soy Broth (TSB) at 37℃.
PCR amplification and DNA Sequencing of ribosome in S. xylosus and tylosin-resistant S. xylosus
Based on the genomes of S. xylosus and tylosin-resistant S. xylosus (tylosin 128µg/mL), the rplC, rplD and rplV fragments were amplified. All primers were listed in Table S3. According to the previous described method in our lab, the genomes of S. xylosus and tylosin-resistant S. xylosus were extracted. We choose the PrimeSTAR GXL DNA polymerase to amplify the fragments of rplC, rplD and rplV. Then, the PCR products were visualized by agarose gel electrophoresis. Furthermore, DNA sequences of PCR products were tested at Comate Bioscience company limited. Subsequently, the DNAMAN software was used to compare amino acid (nucleotide) sequence of S. xylosus with tylosin-resistant S. xylosus.
Construction of the recombinant shuttle plasmids
The shuttle recombinant plasmids (PBT2-rplC, PBT2-rplD and PBT2-rplV) were implemented according to a previous protocol in our lab with some modifications [26] (Figure S2). The upstream and downstream fragment of rplC, rplD and rplV genes were amplified from the genomic DNA of S. xylosus. Meanwhile, the gfp gene and mutant fragments of rplC, rplD and rplV were received from plasmids, including gfp-T, mutant rplC-T, rplD-T and rplV-T. Then, as determinate order, the above PCR products were linked by overlap PCR and cloned to pClone007 Blunt Simple vector. The constructed plasmids were named based on mutant sites as pClone007-rplC, pClone007-rplD and pClone007-rplV. Next, the constructed plasmids were digested and cloned using the restriction sites of the E.coli-Staphylococcus shuttle vector pBT2, thus the recombinant shuttle plasmids PBT2-rplC, PBT2-rplD and PBT2-rplV were received. The above primers and restriction sites used in this section were listed in Table S4.
Construction of the mutant S. xylosus
The mutant strains were created using a previously described protocol [27] with some modifications (Figure S2). The above recombinant shuttle plasmids were introduced into S. xylosus by electroporation. The strains with recombinant shuttle plasmids were grown in TSB at 37 °C for 24 hours, then transferred to fresh medium at 42 °C for another 24 hours, which was identified as one passage. Based on the gfp gene, flow Cytometry (FC) was used to detect the mutant strains of 7, 9 and 11 passages. Meanwhile, the blank strains without recombinant shuttle plasmid served as control. Furthermore, the mutant strains screened by FC were identified by PCR amplification and sequences of mutant fragments (Figure S3). Finally, the mutant strains were named as L3 (rplC) N135G, A137G, S142A and R162K mutant S. xylosus, L4 (rplD) S158N and A164E mutant S.xylosus, L22 (rplV) 97KRTSAIN98 insertion mutant S. xylosus.
Evaluation of mutant S. xylosus susceptibility
Minimal inhibitory concentration (MIC) assays of tylosin and florfenicol to S. xylosus and mutant S. xylosus were done as previously reported [28]. Briefly, S. xylosus and mutant strains were grown overnight at 37 ℃. The overnight cultures were diluted in sterile physiological saline, which correspond to 1 × 108 colony-forming units/mL. Then, the cultures were diluted again with TSB to 1 × 106 colony-forming units/mL. Finally, 100 mL samples were added to the 96-well plate containing serial dilutions of tylosin in culture medium. Control bacterial and medium were cultivated in the absence of tylosin. The MIC was defined as the lowest concentration of inhibitor to visually inhibit growth. The above assays were repeated 3 times.
MD simulation
We employed MD simulations for S. xylosus 23S rRNA, L22 and L22 97KRTSAIN98 insertion mutant to obtain reasonable corresponding three-dimensional structures and study the functional changes in the structure. All MD simulations were performed using GROMACS v2018 package [29]. All systems were solvated in a cubic water box with a 12 Å buffer distance between the solvent box wall and the nearest solute atoms and neutralized by addition of 150 mM Na+ or Cl- ions. AMBER99SB-ILDN force field was assigned to the RNA, protein, and ions [30]. Water was modeled using the TIP3P water model. All structure models were first minimized to relax the solvent and optimize the system using steepest descent. Then, a short 100 ps NVT pre-equilibration simulation was performed whereby the heavy atoms of RNA or protein were positional restrained using a force constant of 200 kJ/(mol•nm). Finally, the production run, containing 23S rRNA and L22, were positional restrained in which to the backbone of ribose (C1’ atom), phosphate (P atom) and Cα atom a ositional force constant of 200 kJ/(mol•nm) was applied. L22 and L22 97KRTSAIN98 insertion mutant production runs were performed without any positional constraints. The temperature of simulations was controlled using the Nose-Hoover thermostat and the pressure was held at 1 bar using an isotropic coupling to the Parrinello-Rahman barostat. Long-range electrostatic interactions were calculated using the particle mesh Ewald method with a real-space cut off value of 1.2 nm. Nonbonded interactions were cut off at 1.2 nm.
Docking
The docking of antibiotics to the receptor was performed using Glide [31]. Tylosin and florfenicol were produced in Schrodinger Maestro software. The LigPrep module in Schrodinger software was introduced for geometric optimization using OPLS_2005 force field. The receptor models were prepared in Schrodinger software under OPLS_2005 force field. Hydrogen atoms were added according to the physiological pH (7.5) with the PROPKA tool in Protein Preparation tool in Maestro to optimize the hydrogen bond network. Constrained energy minimizations were conducted on the full-atomic models. Cubic boxes centered on the tylosin and florfenicol mass center with a radius 12 Å for peptide exit tunnel and PTC of receptor.
Investigation of resistant proteins in the evolution of tylosin-resistant S. xylosus at the mRNA level
Here, five proteins related to resistance were investigated at the mRNA level (16s rRNA was used as an internal control). Moreover, the primers of target genes are listed in Table S5. Total RNA of different levels of tylosin-resistant S. xylosus was extracted as specified by manufacturer, including tylosin (8 µg/mL, 32 µg/mL and 128 µg/mL). Next, equivalent amount of total DNA-free RNA from the last sample was reverse-transcribed using Prim-Script TM RT reagent kit with gDNA Eraser. Finally, the real time PCR program was performed for 40 cycles (95 ℃ for 15 s, 60 ℃ for 35 s) after initial 30 s incubation at 95 ℃. The assays were repeated 3 times.
Investigation of resistant proteins in the evolution of tylosin-resistant S. xylosus at mRNA level
Here, five proteins related to resistance were investigated at the mRNA level (16s rRNA was used as an internal control). Moreover, the primers of target genes are listed in Table S5. Total RNA of different levels of tylosin-resistant S. xylosus was extracted as specified by the manufacturer, including tylosin (8 µg/mL, 32 µg/mL and 128 µg/mL). Next, equivalent amount of total DNA-free RNA from the last sample was reverse-transcribed using Prim-Script TM RT reagent kit with gDNA Eraser. Finally, the real time PCR program was performed for 40 cycles (95 ℃ for 15 s, 60 ℃ for 35 s) after initial 30 s incubation at 95 ℃. The assays were repeated 3 times.
Investigation of resistant proteins in mutant S. xylosus at mRNA level
Here, five proteins related to resistance were investigated at mRNA level. 16S rRNA was used as an internal control. The list of primers is shown in Table S5. Total RNA of S. xylosus and mutant S. xylosus was extracted. Then, the equivalent amount of total DNA-free RNA from the last sample was reverse-transcribed using Prim-Script TM RT reagent kit with gDNA Eraser. Ultimately, the real-time PCR reactions were performed for 40 cycles (95 ℃ for 15 s, 60 ℃ for 35 s) after initial 30 s incubation at 95 ℃. The assays were repeated 3 times.
Statistical analysis
All MIC values were determined in triplicate to ensure reliability. Data on real-time PCR were analyzed (and figures drawn) using GraphPad Prism. DNAMAN and blast was applied to identify the DNA sequence.