The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses

Abstract

Like its homologs throughout the biological world, the L-isoaspartyl protein repair methyltransferase of Escherichia coli, encoded by the pcm gene, can convert abnormal L-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885-9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in rpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158-4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcm mutant lacks these phenotypes. Interestingly, the newly constructed pcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42 degrees C. The pcm mutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functional pcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.

FIG. 1

Stationary-phase survival of pcm mutants and complemented strains. Cultures of the indicated strains were grown in M9 medium for 24 h (day 0), and the number of viable cells remaining was determined at intervals for the next 10 days. The number of CFU is shown as a percentage of the maximum number of CFU; the cultures did not always reach their maximum levels within the first 24 h. Survival of the original pcm deletion mutant strain, CL1010 (––□––) is shown compared to that of its parent, MC1000 (○), to those of strains complemented with a chromosomal wild-type pcm (JV1026) (▪) or rpoS (HC1011) (▴) gene, and to that of a newly constructed pcm deletion mutant strain, JV1068 (—□—).

FIG. 2

Effect of methanol on stationary-phase survival of the pcm mutant strain JV1068. Methanol was added to 0.5% (vol/vol) to cultures grown overnight in LB broth, and the number of viable cells remaining was monitored for 10 days thereafter. The points are shown as the percentage of the day 0 CFU remaining viable and are averages of five trials: the error bars represent 1 standard deviation. The strains used were as follows: the untreated parental strain (untreated MC1000) (○), a methanol-treated parental strain (MC1000) (•), a pcm mutant (JV1068) (□), and a pcm mutant complemented with attB::pcm⁺ (JV1083) (▪).

FIG. 3

Effect of paraquat on stationary-phase survival of the pcm mutant strain JV1068. Paraquat was added to a concentration of 0.1 mg/ml to cultures grown overnight in LB broth, and the number of viable cells remaining was monitored for 10 days thereafter. The points are shown as logs of the percentage of the day 0 CFU remaining viable (averages of at least five trials) for the untreated parental strain (untreated MC1000) (○) and for the paraquat-treated parental strain (MC1000) (•), a pcm mutant (JV1068) (□), and a pcm mutant complemented with attB::pcm⁺ (JV1083) (▪). Viable JV1068 cells remaining on days 8 and 10 were below the limit of detection, which was approximately 50 cells/ml.

FIG. 4

Effects of osmotic and heat stresses on stationary-phase survival of the pcm mutant strain JV1068. (A) Survival curve showing long-term maintenance in LB broth of the untreated parental strain (untreated MC1000) (○) and the effect of adding NaCl to 0.5 M on the parental strain (MC1000) (•), the pcm mutant (JV1068) (□), and the pcm mutant complemented with attB::pcm⁺ (JV1083) (▪). The points shown are averages of six trials; the error bars represent 1 standard deviation. (B) Survival curve for the untreated parental strain (untreated MC1000) (○) and the effect of heating the parental strain (MC1000) (•), the pcm mutant (JV1068) (□), and the complemented pcm mutant (JV1083) (▪) to 42°C for 2 h per day. The points shown are averages of at least three trials; the error bars represent 1 standard deviation.

FIG. 5

Deletion of pcm affects the development of the GASP phenotype. A culture of the competitor strain of interest (open symbols) which had been maintained in stationary phase for 10 days was mixed in a 1:1,000 ratio with a fresh overnight culture of JV1094 (Km^r parental strain; closed symbols). The competitor strains were as follows: (A) aged JV1090 (Tc^r parental strain); (B) aged JV1098 (Tc^r pcm mutant complemented by attB::pcm⁺); (C to E) aged JV1093 (Tc^r pcm mutant). Each panel represents the result of a single experiment, representative of at least six experiments (A and B) or typical of the three different types of outcomes observed for the pcm mutants in eight experiments (C to E). Results similar to those shown in panels C and E were observed twice each, while results similar to those shown in panel D were observed four times.