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, beta-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 lad-based transcriptional reporters. The vectors code for different ribosomal binding sites, allowing for tailored lacZ expression. A Delta crtM::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)(3-)(/4)] 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. IMPORTANCE Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors. Oxygen tension varies greatly at infection sites, and many abscesses are devoid of oxygen. In this study, we have developed novel tools and methods to study how and when S. aureus alters transcription of genes. A key advantage of these methods and tools is that they can be utilized in the presence and absence of oxygen. A better understanding of anaerobic gene expression in S. aureus will provide important insights into the regulation of genes in low-oxygen environments.

Automated summary

Transcriptional reporters are essential tools for studying gene regulation, and in this study, new vectors were developed for markerless insertion of codon-optimized lacZ-based reporters in Staphylococcus aureus. These vectors allow tailored lacZ expression with different ribosomal binding sites. The deletion of crtM gene allows for blue-white screening without interference from carotenoid production, and the methods described are compatible for monitoring transcriptional activity in anaerobic cultures. The study provides important insights into gene regulation in low-oxygen environments for S. aureus, a key human pathogen.