Abstract

Evidence of ageing in the bacterium Escherichia coli was a landmark finding in senescence research, as it suggested that even organisms with morphologically symmetrical fission may have evolved strategies to permit damage accumulation. However, recent work has suggested that ageing is only detectable in this organism in the presence of extrinsic stressors, such as the fluorescent proteins and strong light sources typically used to excite them. Here we combine microfluidics with brightfield microscopy to provide evidence of ageing in E. coli in the absence of these stressors. We report (i) that the doubling time of the lineage of cells that consistently inherits the ‘maternal old pole’ progressively increases with successive rounds of cell division until it reaches an apparent asymptote, and (ii) that the parental cell divides asymmetrically, with the old pole daughter showing a longer doubling time and slower glucose accumulation than the new pole daughter. Notably, these patterns arise without the progressive accumulation or asymmetric partitioning of observable misfolded-protein aggregates, phenomena previously hypothesized to cause the ageing phenotype. Our findings suggest that ageing is part of the naturally occurring ecologically-relevant phenotype of this bacterium and highlight the importance of alternative mechanisms of damage accumulation in this context.This article is part of a discussion meeting issue ‘Single cell ecology’.

Highlights

  • Ageing, or senescence, can be broadly defined as a decline in performance with advancing age

  • Early research on unicellular organisms that exhibit morphologically asymmetric fission revealed that the daughter cell inheriting the older structures of the parental cell shows a longer doubling time than the original cell that produced it

  • Our analyses reveal that the doubling times of cells in the old pole lineage do significantly increase with increasing generation number (LMM: x21 1⁄4 38:327, p < 0.001; controlling for effects of cell polarity, run and channel; electronic supplementary material, table S1); an effect that is readily apparent over the first 11 generations in both runs when inspecting the doubling time data

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Summary

Introduction

Senescence, can be broadly defined as a decline in performance (reproductive success or survival) with advancing age. In order to phenotype new and old pole daughter cells for (i) doubling times, (ii) glucose uptake and (iii) the existence of misfolded protein aggregates, we combined time-lapse microscopy with the microfluidic mother machine device whose fabrication and handling has been previously reported [25,32] This device, made of polydimethylsiloxane (PDMS), is equipped with an array of dead-end microfluidic channels with width and height of 1.5 μm and a length of 25 μm. Upon acquiring each brightfield image the microscope was switched to fluorescent mode, ThT was excited by using a FITC filter and a blue LED (see above) at 20% of its intensity and a fluorescence image was acquired by using an exposure time of 0.03 s These measurements were conducted during separate experimental mother machine runs from the runs that were used to characterize the ageing and asymmetric division phenotypes in §2b,c above (thereby avoiding the use of fluorescent proteins and strong light sources during the latter). Statistical significance of the difference in misfolded protein aggregate formation between wild-type E. coli and GFP expressing or misfolded protein producing E. coli was evaluated via an unpaired t-test with Welch’s correction

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