2 ABSTRACT The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH superfamily that control concentrations of the "alarmones" (p)ppGpp. This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential antibacterial target. Current understanding of RelA mediated responses are based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of Francisella tularensis RelA showed the similarities and differences of this enzyme compared to the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/mL. In contrast to other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp with an EC 50 of 60 ± 1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from Escherichia coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia
