On 13 October at 10:15 Sofia Raquel Alves Oliveira will defend her doctoral thesis “HPLC analysis of bacterial alarmone nucleotide (p)ppGpp and its toxic analogue ppApp”
Professor Tanel Tenson, University of Tartu
Senior Researcher Vasili Hauryliuk, University of Tartu
Prof. Dr. Jörg Stülke, Georg-August-University Göttingen (Germany)
Bacteria through evolution developed numerous adaptation mechanisms that made them survive in harsh environmental conditions. Therefore, to protect themselves from environmental challenges bacteria evolved complex molecular networks that leads to suitable physiological responses by acquiring resistance to antibiotics, forming biofilms or by entering in a dormant state. These adaptation mechanisms depend on enzymatic activity of specific proteins that sense and respond to stress. The responses of these stresses are mediated by synthesis and degradation of signaling molecules that can regulate transcription and protein activities. The PhD work comprise the study of stringent response that is one of the most widely spread adaptive mechanism in bacteria. This mechanism is orchestrated by RelA SpoT Homologue (RSH) enzymes that produce and degrade a highly charged alarmone nucleotide called guanosine(penta)tetraphosphate ((p)ppGpp), comprising guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred as (p)ppGpp. The (p)ppGpp-mediated signaling is one of the master regulators of bacterial physiology and plays an important role in bacterial virulence, and tolerance to antibiotics. In order to quantify the varying levels of (p)ppGpp and housekeeping nucleotides in different stress conditions as well as during normal bacterial growth, we developed a HPLC-based quantification method. Using Escherichia coli and Bacillus subtilis as the two representatives of Gram-negative and Gram-positive bacteria, I studied the effects of antibiotic treatment on the cellular levels of ppGpp, (p)ppGpp as well as housekeeping nucleotides such as ATP and GTP. Finally, using the HPLC-based approach, I discovered that a toxic Small Alarmone Synthetase RSH from Cellulomonas marina, in addition to coproducing ppGpp alarmone synthesizes a highly toxic ppGpp analogue, ppApp. Together with the recent report by Laub and Whitney labs who described Pseudomonas aeruginosa Tas1 – a divergent RSH enzyme that acts as a toxic effector of a secretion system via production of (pp)pApp (Ahmad et al., 2019) this discovery opens up a new direction in studies of RSH enzymes.