Fusion of the N-terminal 119 Amino Acids of RelA with the CTD Domain Render Growth Inhibitory Effects of the Latter, (P)ppgpp-Dependent

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environments and metabolic perturbations. They are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of intracellular (p)ppGpp levels. Characterization of the mutant studied here indicates that moderate level expression of RelA appreciably reduces growth of cells wherein the basal levels of (p)ppGpp are higher than in the wild type without elevating the levels further. Consistent with this result, a large part of the growth inhibition effect is reproduced by overexpression of RelA NTD-CTD fusion lacking the (p)ppGpp synthesis function. A null mutation in relA abolishes this growth inhibitory effect suggesting its requirement for basal level synthesis of (p)ppGpp. Accordingly, increase in the (p)ppGpp levels in the relA1 mutant by spoT202 mutation largely restored the growth inhibitory effects of overexpression of RelA NTD-CTD fusion. Expression of this construct consisting of 119 amino acids of the N-terminal hydrolytic domain (HD) fused in-frame with the CTD domain (±TGS domain) renders the growth inhibitory effects (p)ppGpp-responsive-inhibited growth only of spoT1 and spoT202 relA1 mutants. This finding uncovered an hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function, unraveling the importance of RelA NTD-HD domain for its regulatory role. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes with possible implications in the feedback regulation of the (p)ppGpp synthesis function, a proposal that accounts for the nonlinear kinetics of (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.