4.1. Molecular Biology.

Allelic knockouts of NsaS and the cognate response regulator NsaR were made in the S. aureus Newman background using the knockout strategy via the pIMAY plasmid as described previously. Briefly, plasmid integration is achieved using a temperature sensitive origin of replication, and temporarily confined cultivation in the presence of antibiotic selection under nonpermissive conditions for pIMAY (37 °C). Enrichment of desired doublecrossover candidates was achieved by using an ATc-induced antisense secY RNA based counter-selection that removes single-crossover events. The genomic identity of resulting strains was checked by PCR and sequencing to confirm deletion of the genes. To prevent polar effects, the knockout was designed so that the first 3 and last 3 amino acids of NsaS remained intact. All plasmid transformations into S. aureus Newman were conducted by electroporation (BioRad electro- porator, 1 mm cuvette, 2100 V, 1.1 ms pulse) using ~1 ug of plasmid in 150 μL of competent cells prepared using the sucrose method (reference) with 3 h of recovery in BHI broth. Typical transformation efficiency for the subcloned NsaS-pIMAY plasmids was 10–20 colonies/ug of DNA. Primers and strains are listed in Table S1. For the structural biology, NsaS was cloned out of S. aureus Newman genomic DNA and inserted into the pEXP5 T7-promoter based expression plasmid using a Gibson cloning strategy. The expression of several different constructs and tags was tested. Best results were obtained for a construct that contained the first 86 residues of NsaS followed by a GGCGG and a 6-His tag for purification. The plasmid and protein sequence for this is in the SI. Single cysteine mutations were generated for at 52 positions with the construct of NsaS using a standard Quikchange mutation strategy.