Structural and Mechanistic Analysis of (p)ppGpp Synthetases

The ability of microorganisms to survive under a large variety and rapidly changing environmental conditions is one of their most outstanding features and allowed them to establish within all niches of our planet. To do so, microorganisms have developed a mechanism called the stringent response (SR). The SR relies on the presence of the nucleotide second-messengers (p)ppGpp that contribute to reallocation of resources during stressful environmental conditions. Understanding the broad variety of adaptation processes mediated by the SR therefore necessitates to decipher the metabolism of (p)ppGpp. The stringent factor RelA was long thought to solely account for synthesis and degradation of (p)ppGpp. However, two additional (p)ppGpp synthesizing enzymes, SAS1 and SAS2, were discovered recently. This work presents an in-depth structural and mechanistic characterization of SAS1 and SAS2. Both proteins are subject to allosteric regulation allowing them to integrate different environmental stress stimuli into the framework of the SR. However, SAS1 and SAS2 also mediate adaptation of the microorganism in the absence of environmental stress stimuli, e.g. lack of nutrients. By this, they provide promising targets for the development of future antibiotics guided by the elucidation of their structure and mechanism present in this work. Analysis of (p)ppGpp effecting various cellular targets reveals that the SR confers adaptation processes in a wide intracellular concentration range. This sheds new light on the SR as a mechanism of gradual response to subtle changes in the environment rather than following an ‘all or nothing’ paradigm