Lincomycin

The tulathromycin (TUL), erythromycin (ERY), and lincomycin (LIN) were dissolved in methanol and then diluted with deionized water. The ciprofloxacin (CIP), tetracycline (TET), and rifampicin (RIF) were dissolved in deionized water. Tulathromycin was purchased from Liu He animal Pharmaceutical Co., Ltd. (Qingdao, China). The erythromycin, lincomycin, ciprofloxacin, tetracycline, and rifampicin were purchased from SIGMA.

S. equorum strains cultured in TSB were normalized to 0.5 turbidity at OD₆₀₀ and then diluted 1:100 in TSB supplemented with antibiotics to confirm the function of annotated antibiotic resistance genes. The antibiotics chloramphenicol, ciprofloxacin, erythromycin, lincomycin, methicillin, penicillin G and tetracycline were purchased from Sigma (St. Louis, MO, USA) and employed at concentrations of 30, 5, 15, 30, 8, 6 and 30 μg/ml, respectively, based on previous research results^{11 (link), 13 (link)} and the guidelines of methicillin for S. aureus set out by the Clinical and Laboratory Standards Institute²⁸ . The salt tolerance of S. equorum strains was determined by examining their growth in TSB supplemented with NaCl at concentrations of 15% (w/v), 20% and 25%. To determine the salt tolerance of E. coli transformants, NaCl was employed at a final concentration of 3% or 6% in LB broth. Cell growth was monitored by measuring OD₆₀₀ using a Varioskan Flash (Thermo Scientific, Waltham, MA, USA).

For beta-galactosidase measurements, the B. subtilis JH642-CM21 cells complemented with plasmids containing DesK variants were grown in Spizizen salts (K₂HPO₄, 14.8 g/L, KH₂PO₄, 5.4 g/L, (NH₄)₂SO₄, 2 g/L, tri-Sodium citrate·2H₂O 1.9 g/L, and MgSO₄·7H2O 0.2 g/L), adjusted to different pH with NaOH or HCl and supplemented with glycerol 0.1%, tryptophan 50 μg/mL, phenylalanine 50 μg/mL, Casamino Acids 0.05%, Lincomycin 25%, Erythromycin 0.1%, Chloramphenicol 5 μg/mL, Kanamycin 5 μg/mL, and Spectinomycin 100 μg/mL. All reagents were purchased from Sigma Aldrich with molecular biology purity. β-galactosidase activity was assayed as previously described [9 (link),10 ]. The results shown are the average of five independent experiments. Standard deviations are shown with error bars.

The antimicrobial agents used in this study included tilmicosin, clarithromycin, azithromycin, erythromycin and tiamulin from Fluka Analytical (St. Louis, MO, USA), tylosin from Serva (Heidelberg, Germany) and lincomycin, a lincosamide, from Sigma (St. Louis, MO, USA). The stock antibiotic solutions were sterilized by filtration through 0.2 µm pore size membrane filters (Millipore, Madrid, Spain).

We explored multiple methods to introduce lincomycin into maize. Watering intact seedlings with a lincomycin solution resulted in an unacceptably slow response time. Vacuum infiltration of detached leaves had secondary effects on chloroplast ribosome behavior, as revealed by Ribo-seq analysis of a mock-treated sample. Addition of lincomycin to maize protoplasts completely inhibited D1 synthesis within 10–15 min, but protoplast preparation is time consuming and is unsuitable for studies of dark to light transitions. We ultimately chose to introduce lincomycin into maize seedlings through thread wicks, because chloroplast protein synthesis in leaves was inhibited in 10–15 min (as assayed by pulse-labeling) and seedlings remained intact. Cotton threads (DMC crochet thread size 5) soaked in 1 mg/ml lincomycin (Sigma) were sewn four times through the stem of each seedling beneath the first (lowest) leaf. With 2 threads in each sewing, each wick consisted of 8 threads. The threads were cut at ~5 cm and the ends were placed in a 1.5 ml tube containing lincomycin. The apical half of leaves two and three were processed for ribosome profiling.