Growth curves.

Staphylococcus cultures were grown overnight in TSB at 37°C with shaking at 250 rpm. For aerobic, metal-restricted, and NO-treated bacterial growth curves, overnight cultures of S. aureus were washed twice with phosphate-buffered saline (PBS) and diluted into TSB with or without 2,2-dipyridyl (1 mM) or into CDM with or without carbon to an initial OD at 600 nm (OD₆₆₀) of 0.04. Diluted cultures were then aliquoted into a 96-well plate (200 µl/well) and incubated in a Tecan Infinite M200 microplate reader set to 37°C with 1-mm orbital shaking. Growth was monitored via absorbance at 650 nm every 15 min for 24 h. For NO growth curves, 10 mM NOC-12 (Santa Cruz Biotechnology; catalog no. 202246) and 1 mM DEA NONOate (A. G. Scientific; catalog no. D-1013) were added to the cultures at an OD₆₅₀ of 0.15. To extend the fermentative phase of S. aureus NO-resistant growth, an additional, identical, dose of NO donors was added to each well 1.5 h later. To ensure continued substrate availability during such prolonged NO exposure (S. aureus utilizes carbon inefficiently during NO-induced fermentation), we used 50 mM glucose for these experiments. For anaerobic growth curves, the overnight cultures were washed twice with PBS and diluted into 5 ml of prewarmed (37°C) TSB or CDM with or without carbon with or without 50 mM potassium nitrate to an OD₆₆₀ of 0.08. Cultures were prepared in duplicate in 16- by 150-mm glass tubes containing 1-mm stir bars. Following dilution, cultures were immediately transferred into a Coy anaerobic chamber and grown at 37°C with stirring. Growth was monitored hourly by reading absorbance at 650 nm.