Metabolic adaptation to consume butyrate under prolonged resource exhaustion.

Bacteria must often survive following the exhaustion of their external growth resources. Fitting with this need, many bacterial species that cannot sporulate, can enter a state known as long term stationary phase (LTSP) in which they can persist for years within spent media. Several recent studies have revealed the dynamics of genetic adaptation of Escherichia coli under LTSP. Yet, the metabolic consequences of such genetic adaptation were not addressed. Here, we characterized the metabolic changes LTSP populations experience, over the first 32 days under LTSP. This allowed us to link genetic adaptations observed in a convergent manner across LTSP populations back to their metabolic adaptive effect. Specifically, we demonstrate that through the acquisition of mutations combinations in specific sets of metabolic genes, E. coli acquires the ability to consume the short chain fatty acid butyrate. Intriguingly, this fatty acid is not initially present within the rich media we used in this study. Instead, it is E. coli itself that produces butyrate during its initial growth within fresh rich media. The mutations that enable butyrate consumption allow E. coli to grow on butyrate. However, the clones carrying these mutations rapidly decrease in frequency, once the butyrate is consumed, likely reflecting an associated cost to fitness. Yet despite this, E. coli populations show a remarkable capability of maintaining these genotypes at low frequency, as standing variation. This in turn allows them to more rapidly re-adapt to consume butyrate, once it again becomes available to them.

Density-dependent effects are the main determinants of variation in growth dynamics between closely related bacterial strains.

Although closely related, bacterial strains from the same species show significant diversity in their growth and death dynamics. Yet, our understanding of the relationship between the kinetic parameters that dictate these dynamics is still lacking. Here, we measured the growth and death dynamics of 11 strains of Escherichia coli originating from different hosts and show that the growth patterns are clustered into three major classes with typical growth rates, maximal fold change, and death rates. To infer the underlying phenotypic parameters that govern the dynamics, we developed a phenomenological mathematical model that accounts not only for growth rate and its dependence on resource availability, but also for death rates and density-dependent growth inhibition. We show that density-dependent growth is essential for capturing the variability in growth dynamics between the strains. Indeed, the main parameter determining the dynamics is the typical density at which they slow down their growth, rather than the maximal growth rate or death rate. Moreover, we show that the phenotypic landscape resides within a two-dimensional plane spanned by resource utilization efficiency, death rate, and density-dependent growth inhibition. In this phenotypic plane, we identify three clusters that correspond to the growth pattern classes. Overall, our results reveal the tradeoffs between growth parameters that constrain bacterial adaptation.

Dynamics of Adaptation During Three Years of Evolution Under Long-Term Stationary Phase.

Many bacterial species that cannot sporulate, such as the model bacterium Escherichia coli, can nevertheless survive for years, following exhaustion of external resources, in a state termed long-term stationary phase (LTSP). Here we describe the dynamics of E. coli adaptation during the first three years spent under LTSP. We show that during this time, E. coli continuously adapts genetically through the accumulation of mutations. For nonmutator clones, the majority of mutations accumulated appear to be adaptive under LTSP, reflected in an extremely convergent pattern of mutation accumulation. Despite the rapid and convergent manner in which populations adapt under LTSP, they continue to harbor extensive genetic variation. The dynamics of evolution of mutation rates under LTSP are particularly interesting. The emergence of mutators affects overall mutation accumulation rates as well as the mutational spectra and the ultimate spectrum of adaptive alleles acquired under LTSP. With time, mutators can evolve even higher mutation rates through the acquisition of additional mutation rate-enhancing mutations. Different mutator and nonmutator clones within a single population and time point can display extreme variation in their mutation rates, resulting in differences in both the dynamics of adaptation and their associated deleterious burdens. Despite these differences, clones that vary greatly in their mutation rates tend to coexist within their populations for many years, under LTSP.

Culture Volume Influences the Dynamics of Adaptation under Long-Term Stationary Phase.

Escherichia coli and many other bacterial species, which are incapable of sporulation, can nevertheless survive within resource exhausted media by entering a state termed long-term stationary phase (LTSP). We have previously shown that E. coli populations adapt genetically under LTSP in an extremely convergent manner. Here, we examine how the dynamics of LTSP genetic adaptation are influenced by varying a single parameter of the experiment-culture volume. We find that culture volume affects survival under LTSP, with viable counts decreasing as volumes increase. Across all volumes, mutations accumulate with time, and the majority of mutations accumulated demonstrate signals of being adaptive. However, positive selection appears to affect mutation accumulation more strongly at higher, compared with lower volumes. Finally, we find that several similar genes are likely involved in adaptation across volumes. However, the specific mutations within these genes that contribute to adaptation can vary in a consistent manner. Combined, our results demonstrate how varying a single parameter of an evolutionary experiment can substantially influence the dynamics of observed adaptation.