PCM and survival. In long-term stationary phase, isoaspartyl damage accumulates over time. One might imagine that PCM-deficient bacteria would show impaired survival, as more and more proteins become damaged. As shown at right, however, this is not the case: mutants in which the pcm gene has been deleted (blue line) survive just as well as wild-type (unmutated) cells (black line)¹. In contrast, the green line in the figure shows the result of mutating rpoS, a gene known to be important in stationary-phase survival.

PCM and stress. However, PCM is important in long-term stationary phase survival if the bacteria are also subjected to an environmental stress: oxidative stress (using paraquat as a source of reactive oxygen species), osmotic stress (high salt), methanol or repeated heat stress (1-2 hrs/day at 42°C). The pcm mutant survives less well under these stressful conditions than its wild-type counterpart¹. Survival curves for methanol and paraquat are shown below. The black line shows survival for wild-type cells, the blue line is the pcm mutant, and the red line is the mutant complemented with a functional pcm gene. Open circles show the survival of unstressed cells.

PCM and competition. Even in the absence of stress, PCM-deficient mutants compete poorly with their wild-type counterparts during long-term stationary phase. When wild-type cells are aged for 10 days, mutations are selected for that allow these cells to out-compete "young" cells--even when the aged cells are greatly outnumbered. This is called the GASP (growth advantage in stationary phase) phenotype². Aged cells in which pcm is deleted can sometimes "win" in the competition, but usually either fail to compete effectively or are out-competed by young wild-type cells. This suggests that isoaspartyl accumulation creates problems even for unstressed cells, but that the effect may be too subtle to measure by the survival assay above.

PCM and pH. Interestingly, the stress phenotypes described above are observed only when cells are grown in a rich medium, such as LB broth. As shown at right, pcm mutants show no survival defect when grown under stress in minimal medium buffered to pH7¹. We hypothesized that the survival defects occur when growth on amino acids raises the pH of the growth medium and increases the rate of isoAsp formation. Survival testing in media buffered to specific pHs indicates that this is the case: pcm mutants survive poorly during long-term stationary phase when exposed to both high pH and a denaturing stress, even in minimal medium. Two North Central undergraduates published these results in Microbiology in 2005³.

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¹Visick, J. E., H. Cai, and S. Clarke. 1998. The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses. J. Bacteriol. 180:2623-2629.

²Zambrano, M. M., and R. Kolter. 1996. GASPing for life in stationary phase. Cell 86:181-184.

³Hicks, W. M., M. V. Kotlajich, and J. E. Visick. 2005. Recovery from long-term stationary phase and stress survival in Escherichia coli require the L-isoaspartyl protein carboxyl methyltransferase at alkaline pH. Microbiology 151:2151-2158.