Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and ␣-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

Orthopedic implant-related bacterial infections are associated with high morbidity that may lead to limb amputation and exert significant financial burden on the healthcare system. Staphylococcus aureus is a dominant cause of these infections, and increased incidence of community-associated methicillin-resistant S. aureus (CA-MRSA) is being reported. The ability of S. aureus to attach to the foreign body surface and develop a biofilm is an important determinant of resistance to antibiotic prophylaxis. To gain insight on CA-MRSA biofilm properties, USA300 biofilm maturation and dispersal was examined, and these biofilms were found to exhibit pronounced, quorum-sensing mediated dispersal from a glass surface. For comparison of biofilm maturation on different surface chemistries, USA300 biofilm growth was examined on glass, polycarbonate, and titanium, and minimal differences were apparent in thickness, total biomass, and substratum coverage. Importantly, USA300 biofilms grown on titanium possessed a functional dispersal mechanism, and the dispersed cells regained susceptibility to rifampicin and levofloxacin treatment. The titanium biofilms were also sensitive to proteinase K and DNaseI, suggesting the matrix is composed of proteinaceous material and extracellular DNA. These studies provide new insights on the properties of CA-MRSA biofilms on implant materials, and indicate that quorum-sensing dispersion could be an effective therapeutic strategy. ß

With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and Sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.