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

Abstract

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 (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT 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 ΔspoT 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 ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT 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 ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT 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.

Bacterial persistence is an active σ S stress response to metabolic flux limitation

“…This finding suggests that here rather SpoT (the second E. coli ppGpp synthase) might be responsible for synthesizing ppGpp and for its increased concentration (Fig C). As a deletion of spoT was never achieved without obtaining spontaneous suppressor mutations in the relA gene (Montero et al , ), and the double knockout strain Δ relA Δ spoT cannot grow without certain amino acids (Xiao et al , ), we directly tested the Δ rpoS strain ( rpoS encodes for σ S ). Deleting rpoS in the BW25113 background and in the Δ10 strain both significantly increased the number of cells adapting to fumarate ( t ‐test P ‐value = 0.0025 and 0.0032, respectively) (Fig B) and thus decreased the number of persisters.…”

“…Molecular Systems Biology Bacterial persistence Jakub Leszek Radzikowski et al concentration ( Fig 1C). As a deletion of spoT was never achieved without obtaining spontaneous suppressor mutations in the relA gene (Montero et al, 2014), and the double knockout strain DrelADspoT cannot grow without certain amino acids (Xiao et al, 1991), we directly tested the DrpoS strain (rpoS encodes for r S ).…”

Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance

“…For this, we overexpressed obg in DspoT strains that express relA alleles with non-inactivating mutations, resulting in (p)ppGpp contents reaching 45%-85% of those of wild-type cells (Montero et al, 2014). Overexpressing obg increased persistence in these strains, pointing to the requirement of (p)ppGpp and not SpoT in Obg-mediated persistence ( Figure 4B).…”

“…All DspoT strains used in (B) express relA alleles with non-inactivating mutations, resulting in (p)ppGpp contents reaching 45%-85% of those of wild-type cells (Montero et al, 2014). In all graphs, empty vector controls are shown in gray, and means ± SEM are presented (n R 3).…”

“…Single-gene knockout mutants and a Dppk Dppx double mutant were constructed by l red-mediated gene replacement (Datsenko and Wanner, 2000), using primers listed in Table S1. DrelA and DspoT mutants ( Figures 4A, 4B, 7A, and 7B) were previously described (Montero et al, 2014). E. coli was grown in lysogeny broth (LB).…”