Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus

Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, b-galactosidase (lacZ)-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment, which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized lacZ-based transcriptional reporters. The vectors code for different ribosomal binding sites, allowing for tailored lacZ expression. A DcrtM::kanR deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activities. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system [Fe(CN)₆^32/42] permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture, including an FAD-binding fluorescent protein (EcFbFP), alpha-hemolysin (hla), or lipase (geh). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries.