Slowest possible replicative life at frigid temperatures for yeast

Microbes and animals often inhabit frigid environments. Little is known about the design principles that govern a cell's slow progression in life – how it proliferates, ages and dies – and potential constraints on slowly progressing life. Challenges lie in monitoring slow, intracellular processes and quantifying their combined effects on cells. Here we use budding yeast to quantitatively establish principles that govern cell proliferation and survival at near-freezing temperatures (0C – 5C). By monitoring individual cells for months at near-freezing temperatures, we found small numbers of cells dividing with weeks-long doubling times, while most others died. We found that cells die and cannot divide primarily from having high levels of Reactive Oxygen Species (ROS). Reducing ROS levels or enabling G1 exit with abundant ROS largely prevented deaths and enabled cell divisions with shortened doubling times. These perturbations yielded a comprehensive view of cell's life at near-freezing temperatures that integrated our measurements of single cells' lifespans, days-to-months-long cell-cycle durations, protein-synthesis rates, and genome-wide transcription rates. With a mathematical model, this systems-level view revealed that protein-synthesis dynamics becomes more rate-limiting for cell duplication while mRNA-synthesis dynamics becomes less rate-limiting as temperature approaches 0C. Consequently, the protein-synthesis rate and ROS together establish low-speed and high-speed limits for life at near-freezing temperatures: shortest and longest possible doubling times. Progressing through cell cycle more slowly than the low-speed limit ensures death. This work establishes a quantitative, systems-level foundation for engineering organisms to live in frigid environments and elucidating fundamental limits to slowing down life. Overall design: Data represent time-series to determine genome-wide transcription rates for various conditions. Wild-type yeast was incubated at 1.0C, 5.0C, or 30.0C with or without 250uM GSH added (3 biological replicates for each condition). The synthetic nucleotide 4-thiouracil ("4tU") was added to medium after two weeks of incubation. The abundance of 4tU-labelled mRNA was then quantified over time using next-generation sequencing (“4tU-seq”). The series has 6 timepoints at 1C, 8 time points at 5C, and 5 time points at 30C. Time point 0 is the reference for each condition. A steady-state sample was also included for each condition. All samples contained the same amount of 4tU-labelled spike-in RNA from Schizosaccharomyces pombe to normalize samples over time and across conditions.