Microfluidic Devices for Studying the Development and Aging of Bacteria



 DESCRIPTION (provided by applicant): Recent research reveals that aging acts not only in eukaryotes, but also in bacteria. This revelation challenges decades of evolutionary theory, which held prokaryotes apart as immortal cell lineages immune to aging effects. More current theory suggests that bacterial aging evolved in order to mitigate the consequences of accumulated damage. Through asymmetric segregation of damage upon division, an aging cell experiences deleterious effects via inherited damage, thereby rejuvenating its relatively damage-free counterpart. This model thus argues that selection for damage control drives the concurrent evolution of asymmetric reproduction and aging, and that aging should evolve as a common life history strategy. Recent observations find aging even in bacteria with morphologically symmetric division, indicating that underlying reproductive asymmetry may indeed occur in essentially all cellular organisms. We hypothesize that asymmetric reproduction in bacteria specifically drives aging via progressive accumulation of damage in aging cells. We will examine aging in the predominant model for asymmetric bacteria, Caulobacter crescentus and various asymmetry mutants. Epifluorescence microscopy will monitor protein aggregation and oxidative damage alongside measures of cell vitality, thereby exploring the proximal mechanism of bacterial aging effects. Varying damage rates using antibiotics and an oxidative stressor will evaluate the ability of specific mutants to segregate and tolerate accumulated damage. We predict that disrupting normal reproductive asymmetry will attenuate the aging process, while at the same time reducing population-wide fitness and increasing susceptibility to damage. Testing the hypothesized link between damage, aging, and asymmetry proves particularly difficult with existing methodology. Aging manifests itself as a decline in vital life history parameters (e.g., cell elongation rate, division rate, and survival) of old cells compared o their rejuvenated counterparts. Aging studies thus require detailed measurements of many cells and their lineages over multiple generations in order to achieve adequate statistical power. We are therefore developing microfluidic devices with integrated nanochannel arrays that permit observation of aging bacterial lineages over extended time periods. The nanochannels constrain growth of the bacteria along a single dimension, and microfluidic channels on each end of the nanochannels direct constant input of fresh media while washing away cells that grow beyond the nanochannels. Such a design permits direct, high-resolution observation of the youngest bacteria (enriched at the nanochannel center) and their immediate descendants over several generations - the key cells for aging studies. Although focused on Caulobacter for our initial studies, this microfluidic device design serves as a general platform to study aging and other epigenetic phenomena in diverse bacteria. The specific aims for the project are to measure aging and its effectors in C. crescentus, explore the connection between asymmetry and aging, and determine the plasticity of bacterial damage control strategies.