The effect of cell death on DNA-replication-based estimates of microbial population growth

Inferring bacterial growth rates is fundamental to understanding microbial interactions and community dynamics, but remains difficult in natural settings where timepoints are limited or organisms are unculturable. In these cases, a widely used method is the origin-to-terminus ratio, or peak-to-trough ratio (PTR), which estimates DNA replication activity by comparing the copy number of DNA at the replication origin and terminus. While PTR correlates well with cellular growth in uniform, idealized environments, it measures replication rather than net growth rate, and thus reflects growth only when there is no cell death. Despite this, PTR is widely applied across a range of laboratory and environmental contexts, where microbial populations frequently experience fluctuating stress, mortality, and subpopulation heterogeneity. Given its widespread use in such settings, we developed a stochastic, cell-based model that explicitly tracks DNA replication and cell death to quantify how different patterns and levels of mortality affect the relationship between PTR and net growth rate. We found that PTR and net growth rate are tightly correlated in idealized conditions; however, systematic deviations emerge when death rates vary over time or across subpopulations. We experimentally validated these predictions by exposing Escherichia coli to osmotic shock or antibiotics, and measuring net growth rate (by spot plating and observing the change in colony counts over time) and DNA replication activity (from qPCR with primers for the origin and terminus). Consistent with the predictions from our model, PTR correlated strongly with net growth rate in standard rich media, but not under stress. Together, these results provide a mechanistic and quantitative framework that clarifies the biological conditions under which PTR can be interpreted as a proxy for net growth rate.