Real-time qRT-PCR.

S. aureus COL was grown in 50 ml of CDM plus 25 mM glucose or Casamino Acids (0.5%) in 250-ml flasks at 37°C with shaking at 250 rpm. At an OD₆₆₀ of 0.5, 25 ml of each culture was added to an equal volume of ice-cold ethanol-acetone (1:1) and frozen at −80°C (aerobic cultures). To assess gene expression during NO exposure, a separate set of cultures (CDM plus glucose) was treated with 5 mM DETA-NONOate (Cayman Chemical; catalog no. 82120) for 1 h, quenched, and then frozen. Lastly, S. aureus COL was grown in 50 ml of CDM plus glucose in the anaerobic chamber at 37°C with stirring. At an OD₆₆₀ of 0.5, 25 ml of culture was removed from the chamber in a 50-ml conical tube devoid of oxygen, immediately quenched, and then frozen. RNA was then harvested, and gene expression was analyzed as previously described (49). Transcript levels of selected genes were normalized to rpoD transcript levels, which deviated very little across our experimental conditions. For the primers used for quantitative reverse transcriptase PCR (qRT-PCR) analysis, see Table S3 in the supplemental material.