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, thel-isoaspartyl protein repair methyltransferase ofEscherichia 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 inrpoS, 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 pcmmutant lacks these phenotypes. Interestingly, the newly constructedpcm 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°C. The pcmmutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functionalpcm 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.

Mutations in the Escherichia coli surE gene increase isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stress-survival phenotypes

“…Strain MC1000 (V 3 e14 3 araD139 v(araAleu)7697 galE15 galK16 v(codB-lac)3 rpsL150 mcrB1 relA1 spoT1) was used as the parent strain for these experiments. The construction of strains JV1068 (MC1000 vpcm: :Cm [5]) and JKI2010 (MC1000 surE: :Km rpoS396 [10,11]) has been described. A surE: :Cm derivative, JV1066, was constructed by inserting a chloramphenicol resistance (Cm ) marker into the BstEII site near the midpoint of surE, with the marker gene oriented in the same direction as surE and pcm.…”

“…A surE: :Cm derivative, JV1066, was constructed by inserting a chloramphenicol resistance (Cm ) marker into the BstEII site near the midpoint of surE, with the marker gene oriented in the same direction as surE and pcm. Chromosomal gene replacement was then carried out using pKAS46 as described previously [5,12]. Strain JV1070, a surE: : Km vpcm: :Cm double mutant, was made by P1 transduction of surE: :Km from JKI2010 into JV1068.…”

“…Activity of the L-isoaspartyl methyltransferase in extracts of stationary-phase E. coli prepared by gentle sonication was determined as described [5] by quantitation of base-labile IR C-methyl esters transferred to an isoaspartyl-containing peptide substrate. Activity of HPII catalase, used as a reporter for RpoS, was measured spectrophotometrically in extracts in which HPI catalase had been heat-inactivated [11].…”

“…After incubation for 2 h at 37³C, the tube was spun brie£y, frozen in dry ice to stop the reaction, rapidly thawed and mixed with 40 Wl of 0.2 M NaOH and 1% SDS. A 60-Wl aliquot was then spotted onto a piece of ¢lter paper, and volatile methyl esters were quantitated as for the methyltransferase assay [5].…”

“…Isoaspartyl residues are recognized and methylated by L-isoaspartyl protein carboxyl methyltransferase (EC 2.1.1.77), resulting in their net repair to normal aspartyl residues in vitro [4]. In the absence of this repair enzyme, detrimental e¡ects have been observed in vivo in Escherichia coli [5], nematodes [6], and mice [7].…”