ABSTRACTThel-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival ofEscherichia coliunder stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identifiedpcmas a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcmmutant produced persisters for an extended period in stationary phase, and a ΔglpDmutation drastically increased persisters in a Δpcmbackground, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than thepcmmutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates inglpDmutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the ΔpcmΔglpDmutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcmmutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage.IMPORTANCEProtein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population inEscherichia colibacteria increased, especially if the metabolic geneglpDwas also mutated. High levels of persisters inpcm glpDdouble mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.
Existence of log-phase Escherichia coli persisters and lasting memory of a starvation pulse
