Synthesis of guanosine 5′-triphosphate,3′ -diphosphate in a spoT strain of Escherichia coli

Abstract

A spoT stringent strain of Escherichia coli rapidly accumulates guanosine 5′-triphosphate,3′-diphosphate (pppGpp) immediately after the onset of isoleucine starvation. Subsequently, its level rapidly falls, as guanosine 5′-diphosphate,3′-diphosphate (ppGpp) continues to rise to the maximum value, which is abnormally high compared with that in the spoT+ strain. The ppGpp level in the spoT strain never reaches a steady state as it does in the spoT+ strain. Immediately after starvation, pppGpp and ppGpp are labeled with [3H]guanosine at a similar differential rate in both the spoT and spoT+ strains, suggesting that the two strains synthesize these nucleotides by the same pathway. However, by 15 min after starvation, the synthesis of these nucleotides is nearly halted in the spoT strain, and is greatly reduced in the spoT+ strain. Since ppGpp is labeled with [3H]guanosine more slowly than pppGpp in the starved spoT+ strain, ppGpp cannot be a precursor of pppGpp. The kinetics of the GTP level during starvation suggests that GTP is a precursor of pppGpp. The observed differences between the spoT and spoT+ strains can be explained by postulating, firstly, that ppGpp negatively controls the conversion of GTP to pppGpp, which is subsequently converted to ppGpp; secondly, that a catabolite of ppGpp negatively controls the conversion of pppGpp to ppGpp; and thirdly, that the spoT mutation primarily reduces the rate of ppGpp catabolism.