Dl serine hydroxamate

Cells were grown at 37°C in LB (10 g/liter NaCl, 10 g/liter tryptone, and 5 g/liter yeast extract), M9 glycerol-Casamino Acids medium (1× M9 salts, 1 mM MgSO₄, 100 μM CaCl₂, 0.4% glycerol, 0.2% Casamino Acids), or M9GAV medium (1× M9 salts, 0.4% glucose, 0.25% each of l-serine and l-threonine, 0.0375% each of l-asparagine and l-glutamine, 0.015% each of all 16 other natural amino acids, 0.2 mM nucleobases, and 1× Kao & Michayluk vitamins) and supplemented with antibiotics as noted at the following concentrations (liquid/plate): carbenicillin, 50/100 μg/ml; kanamycin, 30/50 μg/ml; chloramphenicol, 20/30 μg/ml; and spectinomycin, 50/50 μg/ml. IPTG (100 μM), arabinose (0.2%), or anhydrotetracyline (aTc [100 nM]) was added as an inducer of gene expression unless otherwise noted. For M9GAV experiments, RelA′ was induced with 50 μM IPTG (WT) or 150 μM (RNAP 1⁻2⁻) to achieve comparable induction levels (15 (link)). Plates contained 1.2% agar. dl-Serine hydroxamate (Sigma) was added to cells at a concentration of 1 mg/ml. For rifampin runouts, rifampin (300 μg/μl) and cephalexin (12 μg/μl) were added for 4 h.

In this study, we used two urogenital clinical isolates of N. gonorrhoeae from female patients, named, respectively, T9 (a serotype 9 strain), and T2 (a serotype 2 strain; Martin et al., 1986 (link)). These strains were grown, from frozen stocks (−80°C), on GC agar base (OXOID) supplemented with 1% (v/v) Polyvitox (OXOID) at 37°C in a 5% CO₂ incubator. The liquid medium used for growth was GC broth whose composition (per liter) is as follows: 15 g proteose peptone, 0.5 g corn starch, 4 g K₂HPO₄, 1 g KH₂PO₄, 5 g NaCl, 1% (v/v) Polyvitox (OXOID).
Thiostrepton (MIC 0.9 μg/mL; MBC 3.75 μg/mL), ampicillin (MIC 0.6 μg/mL; MBC 1.25 μg/mL), gentamicin (MIC 3.75 μg/mL; MBC 7.5 μg/mL), nalidixic acid (MIC 1.875 μg/mL; MBC 3.35 μg/mL), rifampicin (MIC 0.23 μg/mL; MBC 0.9 μg/mL), tetracycline (MIC 0.05 μg/mL; MBC 0.8 μg/mL), and DL-serine hydroxamate (MIC 1 mg/mL; MBC 4 mg/mL; all provided by Sigma-Aldrich) were used at the required concentration (values in brackets) in GC broth. Liquid cultures in GC broth were incubated at 37°C on rotary shaker at 200 rpm.