Anhydrotetracycline

The strains and plasmids used in this study are listed in Table S1. The methicillin-resistant S. aureus (MRSA) USA300 strain JE2 (GenBank CP000255) was used throughout the study. The deletion mutant of hflX (SAUSA300_1198) has been described previously (31 (link)). Unless otherwise noted, S. aureus cells were grown at 37 °C in tryptic soy broth (TSB, Difco) at a 5:1 tube- or flask-to-medium ratio with a 1:100 dilution of an overnight seed culture. TSB agar plates were prepared using Difco^TM agar (Difco, BD 281230), Eiken agar (Eiken, E-MJ00), Gelrite (RPI, G35020), and agarose (GenMate, E-3120). When necessary, erythromycin, chloramphenicol, cadmium chloride, kanamycin, and anhydrotetracycline (all from Sigma-Aldrich) were used at 5 μg/ml, 10 μg/ml, 0.15 mm, 75 μg/ml, and 400 ng/ml, respectively. The restriction-deficient S. aureus RN4220 strain was used as a plasmid passage surrogate before the plasmid was transformed into the destination JE2 derivatives. E. coli cells harboring expression vectors were grown at 16 or 37 °C in LB (Difco). Antibiotics were used at 50 (kanamycin) or 100 μg/ml (ampicillin, Sigma-Aldrich). All GTP analogs were from Sigma-Aldrich. Primers were purchased from IDT DNA and are listed in Table S2.

All strains used for MIC determination have been published previously.⁶ (link) In short, the candidate B1 MBL genes bla_MYO-1, bla_ECV-1 and bla_SHD-1 were synthesized and sub-cloned into the pZE21-MSC1 vector (Expressys, Ruelzheim, Germany). Recombinant plasmids were transformed into E. coli C600Z1 (Expressys).⁶ (link)^,28 (link) For MIC determination, single colonies were incubated overnight on Mueller–Hinton II agar (Becton Dickinson, Franklin Lakes, USA) containing 25 mg/L kanamycin and subsequently suspended in 0.85% saline to a cell density with a turbidity equivalent to that of a 0.5 McFarland standard (1.5 × 10⁷ cells/mL). The McFarland solution was uniformly dispersed with a swab onto the agar plates containing 100 ng/mL anhydrotetracycline (Sigma–Aldrich, St Louis, MO, USA). Gradient diffusion strips (Liofilchem, Roseto degli Abruzzi, Italy) were applied and the MICs were determined after 19 h of incubation at 37°C.

Plasmodium falciparum lines MRA 150 (Dd2), MRA 1000 (NF54), and MRA 1238 were obtained from the Malaria Research and Reference Reagent Resource Center (MR4, BEI Resources). The ATG8 conditional TetR-Dozi knockdown line was a generous gift from Ellen Yeh (Stanford University, Stanford, CA). Plasmodium cultures were maintained in A+ human erythrocytes (Valley Biomedical, Winchester, VA) at 3% hematocrit in RPMI 1640 HEPES (Sigma-Aldrich, St. Louis, MO) supplemented with 0.5% Albumax II Lipid-Rich BSA (ALB, Sigma-Aldrich, St. Louis, MO) and 50 mg/L hypoxanthine (Thermo Fisher Scientific, Waltham, MA). This formulation is referred to here as “standard media.” ATG8 TetR-DOZI parasites were maintained with 0.5 μM anhydrotetracycline (Sigma-Aldrich, St. Louis, MO) or supplemented with isopentyl pyrophosphate (Isoprenoids, LC, Tampa, FL), as previously described (20 (link)). Cultures were grown at 37°C and individually flushed with 5% oxygen, 5% carbon dioxide, and 90% nitrogen gas. Dilution of cultures with uninfected erythrocytes and changing of culture medium were performed every other day. Parasitemia was determined by flow cytometry using SYBR Green I staining and kept below 2% during maintenance. Cultures were confirmed negative for mycoplasma approximately monthly using a LookOut Mycoplasma PCR detection kit (Sigma-Aldrich, St. Louis, MO).