(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair.

UNLABELLED: The l-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 of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcm mutant produced persisters for an extended period in stationary phase, and a ΔglpD mutation drastically increased persisters in a Δpcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant 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 in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δpcm ΔglpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcm mutant 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. IMPORTANCE: Protein 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 in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double 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.

Recovery from long-term stationary phase and stress survival in Escherichia coli require the L-isoaspartyl protein carboxyl methyltransferase at alkaline pH.

The L-isoaspartyl protein carboxyl methyltransferase (pcm) can stimulate repair of isoaspartyl residues arising spontaneously in proteins to normal L-aspartyl residues. PCM is needed in Escherichia coli for maximal long-term survival when exposed to oxidative stress, osmotic stress, repeated heat stress or methanol. The effect of pH on a pcm mutant during long-term stationary phase was examined. PCM was not required for long-term survival of E. coli subjected to pH stress alone; however, PCM-deficient cells showed impaired resistance to paraquat and methanol only at elevated pH. The mutant also showed stress-survival phenotypes in minimal medium buffered to pH 9.0. Accumulation of isoaspartyl residues was accelerated at pH 8.0 or 9.0 in vivo, though PCM-deficient cells did not show higher levels of damage. However, the pcm mutant displayed an extended lag phase in recovering from stationary phase at pH 9.0. Protein repair by PCM thus plays a key role in long-term stress survival only at alkaline pH in E. coli, and it may function primarily to repair damage in cells that are recovering from nutrient limitation and in those cells that are able to divide during long-term stationary phase.