Understanding the Limits on Exponential Growth of Bacteria Through Long Term Evolution

Abstract

Bacteria have evolved to survive periods of harsh conditions and to grow and divide rapidly under periods of rich conditions. The shortest sustainable doubling times observed for Escherichia coli are about 17 minutes and other bacteria can steadily sustain doubling times as short as 12 minutes. This appears remarkably fast given the essential processes of energy production, protein synthesis and DNA replication, particularly the time it takes a ribosome to make the ribosomal proteins for another ribosome. The goal of this thesis is to investigate what allows bacteria to double that fast, by asking how and to what extent they can evolve even faster if placed under sustained selective pressure to maximize growth rates. To this end, we built a series of highly automated turbidostats that can maintain bacterial cultures in constant exponential phase growth for tens of thousands of generations under optimal growth conditions, without frequent interruptions or contaminations even in the absence of antibiotics. We have run, and continue to run, what to our knowledge is the longest known continuous culturing evolution experiment. We started with 8 parallel wild-type E. coli populations, and so far ran over 30,000 generations. Despite systematic optimization of the growth conditions, all cultures gradually evolved significantly shorter doubling rates, without any sign of approaching a limit. Populations genetically diverged through accumulating mutations in diverse pathways with complex epistasis. We identified a set of key genes and mutations that potentially are responsible for the higher growth rates. I hope the biological results will help us understand limits on bacterial growth, and that the automated platforms for ultra-long-term evolution experiments will mark the beginning of a million-generation experiment.