Project description:Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity on the ribosome remain elusive. Here, we show a novel role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using random mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wildtype RelA. Our data provide strong evidence to support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.
Project description:Effect of ppGpp on transcript level after RelA overexpression (90 min). The cells are constructed in our lab. They are knock-out mutants in the production of ppGpp(guanosine tetraphosphate) of Legionella, complemented with an over-expression vector containing the relA gene to express the RelA protein (responsible for the ppGpp production). The aim is to follow the gene response in Legionella pneumophila after induction of ppGpp at different time points.
Project description:Growth curves and (p)ppGpp accumulation assays showed that RelA inactivation could influence S. suis growth and led to incapacity of (p)ppGpp synthesis during glucose starvation. To identify the roles of RelA/(p)ppGpp in global gene regulation in S. suis, we compared the transcriptional profiles of SC-19 [a (p)ppGpp+ strain] and ΔrelA [a (p)ppGpp0 strain during glucose starvation] in both glucose-abundant and -deficient CDM in exponential phase by microarray analysis. A less stringent cut-off limit, 2-fold change, was used. qRT-PCR validation displayed the same trends observed in the microarrays
Project description:Growth curves and (p)ppGpp accumulation assays showed that RelA inactivation could influence S. suis growth and led to incapacity of (p)ppGpp synthesis during glucose starvation. To identify the roles of RelA/(p)ppGpp in global gene regulation in S. suis, we compared the transcriptional profiles of SC-19 [a (p)ppGpp+ strain] and ΔrelA [a (p)ppGpp0 strain during glucose starvation] in both glucose-abundant and -deficient CDM in exponential phase by microarray analysis. A less stringent cut-off limit, 2-fold change, was used. qRT-PCR validation displayed the same trends observed in the microarrays relA mutant strain and its parents strain SC-19 were cultured in both glucose-abundant CDM (CDM containing 1% glucose) and glucose-deficient CDM (CDM containing 2% glucose) respectively. The bacteriain in exponential phase were collected for microarray analysis. Three independent experiments were performed.
Project description:DarB is a cyclic di-AMP binding protein consisting of two CBS (Cystathionine-beta synthase) domains. To reveal the global role of the second-messenger, understanding the function of the receptor proteins is fundamental. In order to do so, we aimed to identify potential binding partners of DarB in vitro with an unbiased protein pulldown experiment. In this study, we identified the (p)ppGpp synthetase/ hydrolase RelA as the binding partner of DarB and could confirm these findings with an in vivo interaction experiment.
Project description:Effect of ppGpp on transcript level after RelA overexpression (45 min). The cells are constructed in our lab. They are knock-out mutants in the production of ppGpp(guanosine tetraphosphate) of Legionella, complemented with an over-expression vector containing the relA gene to express the RelA protein (responsible for the ppGpp production). The aim is to follow the gene response in Legionella pneumophila after induction of ppGpp at different time points.
Project description:ppGpp synthesis was activated in exponentially growing cells of Streptomyces coelicolor in the absence of amino acid starvation by using a strain (M653 [DrelA tipAp::relA(1.46kb)]) that expresses a truncated portion of relA under the control of a thiostrepton-inducible promoter. Samples were harvested at 30 min intervals following induction for comparison to a control set of samples from aliquots of the same cultures that were not induced.
Project description:Escherichia coli RelA is a ribosomal factor with strong (p)ppGpp synthesis activity that is dramatically activated in the presence of deacylated tRNA in the ribosomal A-site. RelA is a unique enzyme in that it is directly positively regulated by its product, alarmone nucleotide (p)ppGpp. Using HDX-MS, mulecular docking, biochemsitry, and microbiology approaches, we localise the (p)ppGpp binding site of E. coli RelA and uncover the molecular mechanism of RelA regulation by the alarmone.