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

Abstract

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 ⌬pcm mutant produced persisters for an extended period in stationary phase, and a ⌬glpD mutation drastically increased persisters in a ⌬pcm 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 ⌬pcm ⌬glpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the ⌬pcm 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. 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. PCMdirected 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. I soaspartyl damage to proteins occurs when aspartyl or asparaginyl residues spontaneously isomerize via a succinimide intermediate (1) (Fig. 1), adversely affecting protein function (2). The damage can be repaired when the abnormal isoaspartyl (isoAsp) residue is recognized and methylated by the L-isoaspartyl protein carboxyl methyltransferase (PCM) (Fig. 1)...

The (p)ppGpp synthetase Rsh promotes rifampicin tolerant persister cell formation in Brucella abortus by regulating the type II toxin-antitoxin module mbcTA

“…The relationships between the stringent response alarmone (p)ppGpp and persistence are dissimilar in different bacteria ( Conlon et al, 2016 ; VandenBerg et al, 2016 ; Pacios et al, 2020 ). Previous research from our laboratory indicated that B. abortus was capable of forming multi-drug tolerant persister cells ( Liu et al, 2023 ) .…”

“…These data provide new clues for discovering potential drug targets for clearing Brucella persister cells, as well as for forming new strategies for prevention of Brucella infection. The relationships between the stringent response alarmone (p) ppGpp and persistence are dissimilar in different bacteria (Conlon et al, 2016;VandenBerg et al, 2016;Pacios et al, 2020). Previous research from our laboratory indicated that B. abortus was capable of forming multi-drug tolerant persister cells (Liu et al, 2023).…”

Functional divergence of annotated l-isoaspartate O-methyltransferases in an α-proteobacterium

“…A recent study showed that an E. coli PIMT mutant formed a larger percentage of quiescent, antibiotic-resistant, persister cells than the WT. The authors suggest that in situations where cells cannot repair isoDs, they may respond by becoming persisters, and this may protect cells from the consequences of protein damage (30). This still begs the question of how isoD protein damage might trigger persister formation.…”