Genetic Manipulation of Glycogen Allocation Affects Replicative Lifespan in E. coli.
Bottom Line: We found that certain changes in the regulation of the carbohydrate metabolism can affect aging.These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles.This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported.
Affiliation: Biozentrum, University of Basel, Switzerland.
In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported.
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Mentions: How could the accumulation of glycogen at the old cell pole lead to the termination of growth and division of the cell carrying this pole? Previous studies reported mutual spatial exclusion between (protein) aggregates and the bacterial chromosome [27, 28], raising the possibility that the large polar aggregates in the csrA mutant could lead to the exclusion of the chromosome. To test whether spatial exclusion of the chromosome by the observed glycogen accumulation leads to the short replicative lifespan in this strain, we followed the localization of a fusion protein of Hns to GFP. Hns is a protein that binds to AT-rich regions on DNA , and can thus be used as a marker for the location of the chromosome if fused to GFP. Hns-GFP was excluded from older pole cells in the csrA mutant, and this exclusion became apparent already at early pole ages (Fig 4A, S5 Movie). After the last division, no visible Hns-GFP signal remained in the old pole daughter cell, indicating a loss of chromosomal DNA (Fig 4A and 4B). We measured Hns-GFP intensity in mother and daughter cells of first, second last, and last divisions (Fig 4B), and found a drop in fluorescence in mother cells at the last division, indicating a loss of the chromosome. We scrutinized this finding using a DNA stain; again, after the last division, the old pole daughter cells did not contain visibly stained DNA (S9 Fig). These results thus indicate that the glycogen accumulating at the old pole in cells of the csrA mutant excludes the chromosome, which would explain why these cells then stop growing and dividing. Bacterial cells without chromosomes have been observed before, most prominently in the case of ‘minicells’ ; these minicells are produced as a consequence of mutations in cell division genes , they are about 10 times smaller than normal cells , and there is (as far as we know) no evidence that the loss of the chromosome is specific for cells with old cell poles. In contrast, the csrA cells lose their chromosome ultimately as a consequence of a metabolic change, these cells are only slightly smaller than other cells in the population (S2 Fig), and the loss of the chromosome is specific for cells with old cell poles. While these two observations share the aspect of chromosome deficiency, they are thus different in a number of other aspects.