Synthesis of guanosine 5'-triphosphate,3'-diphosphate in a spo T strain of Escherichia coli

Abstract

A spo T 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 spo T+ strain. The ppGpp level in the spo T strain never reaches a steady state as it does in the spo T+ strain. Immediately after starvation, pppGpp and ppGpp are labeled with [3H]guanosine at a similar differential rate in both the spo T and spo T+ 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 spo T strain, and is greatly reduced in the spo T+ strain. Since ppGpp is labeled with [3H]guanosine more slowly than pppGpp in the starved spo T+ 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 spo T and spo T+ 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 spo T mutation primarily reduces the rate of ppGpp catabolism.