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In  Escherichia coli,  ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of  spoT  null (Δ spoT ) mutants obtained by transducing a Δ spoT  allele from Δ relA Δ spoT  double mutants into  relA +   cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 Δ spoT  clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in  relA  and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of Δ spoT  with  relA  proficient alleles has generally been considered synthetically lethal, we found that 11 Δ spoT  clones displaying high glycogen phenotypes possessed  relA  mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the Δ spoT  clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the Δ spoT  clones revealed that suppressor mutations were restricted to the  relA  locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in  relA , but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the  in   vivo  regulation of  E. coli  RelA ppGpp synthetase.

plos one

Systematic Production of Inactivating and Non-Inactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli