Metabolic adaptation to consume butyrate under prolonged resource exhaustion

Abstract

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.

Escherichia coliadaptation under prolonged resource exhaustion is characterized by extreme parallelism and frequent historical contingency

“…No other lineages ever included any such mutator clones. Interestingly, the pattern by which mutators occur only on the background of lineages established by the RpoB E1272G mutation was also recapitulated in a second study, in which six additional LTSP populations were established [37]. Two additional examples of loci that are mutated predominantly within lineages defined by a specific RNAPC mutation are: (1) the emergence of mutations in both the dctA dicarboxylate transport gene and its promoter on the background of three out of five lineages defined by the RpoC K334Q mutation, and never on the background of any other lineage-defining mutations; and (2) the emergence of specific mutations within the gene dppA and the gcvT promoter as well as different nonsense and frameshift mutations to proQ that occur in four of five lineages defined by the RpoC T428S mutation (the fifth RpoC T428S lineage that did not acquire these mutations was very short-lived, due to the RpoB E1272G-muator lineage overtaking its population by day 42 of the experiment).…”

“…We identified two putative contingencies for which we already know the biological meaning from our previous studies [28,37]. The first contingency involves mutations within the gene dnaQ , which encodes a proofreading component of the DNA polymerase.…”

“…Such mutations occur exclusively on the background of mutations to the gene encoding FadR - the E. coli master regulator of fatty acid metabolism. We have previously shown that this mutation combination allows E. coli to metabolize the short chain fatty acid butyrate, which E. coli itself produces during the first 24 hours of growth within fresh LB, but which wildtype E. coli cannot metabolize [37].…”

Pseudomonas Putida Dynamics of Adaptation under Prolonged Resource Exhaustion

“…Contrary to the impression given by the word 'stationary', bacterial populations do not remain stagnant during LTSP. Instead, they continuously undergo physiological, metabolic and genetic changes, even years into their time under resource exhaustion (Finkel 2006;Avrani, et al 2017;Katz, et al 2021;Ratib, et al 2021;Katz, et al 2023).…”

“…Although the ability to enter LTSP is not limited to a single bacterial species (Lostroh and Voyles 2010;Bruno and Freitag 2011;Reichert, et al 2013;Djebbi-Simmons, et al 2019;Shoemaker, et al 2021), the majority of studies on LTSP have been performed on the model bacterium Escherichia coli Finkel and Kolter 1999;Farrell and Finkel 2003;Finkel 2006;Finkel 2014, 2015;Avrani, et al 2017;Gross, et al 2020;Nandy, et al 2020;Katz, et al 2021;Ratib, et al 2021;Katz, et al 2023) When E. coli is placed into rich media, a short period of exponential growth is followed by a period of decline (often referred to as death phase), after which bacteria enter LTSP. The dynamics of E. coli LTSP adaptation were explored in a recent evolutionary experiment, in which five populations of E. coli were established in July 2015, allowed to enter LTSP and have been maintained under LTSP ever since (Avrani, et al 2017;Katz, et al 2021).…”