Functional characterisation of (p)ppGpp synthetases in Staphylococcus aureus

Staphylococcus aureus is a Gram positive bacterium that can colonise the nose and skin asymptomatically but can also invade the host as a pathogen. In the host, bacteria encounter various nutrient stresses which trigger the activation of the stringent response. This conserved mechanism allows bacteria adapt to environmental changes such as amino acid starvation and cell wall stress. It is mediated by two nucleotides, ppGpp and pppGpp, collectively known as (p)ppGpp. These small alarmones have many binding targets and cause the cells to enter a slow growing state through altering the transcriptional profile and inhibiting ribosome formation. In S. aureus, (p)ppGpp is synthesised by three members of the RSH superfamily: RelSa, RelP and RelQ. In this study we investigate how these synthetases are regulated transcriptionally and post-translationally, as well as the role of the stringent response in stress survival and growth in S. aureus. Promoter-lacZ reporter fusions were used to show that the synthetase genes are negatively regulated by several transcription factors, highlighting the intricacies of their regulation. We have also identified several binding partners of the RelSa and RelP that may play a role in post-translational regulation of the proteins. In particular, we examine the effects of the interaction between RelSa and the arginase, RocF which appears to be augmented by ppGpp. The arginase activity of RocF and the hydrolase activity of RelSa are not affected by the interaction. RelSa was also shown to bind to L-arg, suggesting that non-protein ligands may also regulate RelSa activity. Finally, as arginine metabolism and urease production in S. aureus are regulated by the carbon catabolite repressor CcpA, we investigate the importance of the stringent response in certain aspects of CcpA-regulated metabolism, including long term survival, arginase activity and urease activity using a panel of synthetase mutants.