This content will become publicly available on October 1, 2025
Understanding the interplay between genotype and fitness is a core question in evolutionary biology. Here, we address this challenge in the context of microbial adaptation to environmental stressors. This study explores the role of epistasis in bacterial adaptation by examining genetic and phenotypic changes in silver-adapted Escherichia coli populations, focusing on the role of beneficial mutations in two-component response systems (TCRS). To do this, we measured 24-hour growth assays and conducted whole-genome DNA and RNA sequencing on E. coli mutants that confer resistance to ionic silver. We showed recently that the R15L cusS mutation is central to silver resistance, primarily through upregulation of the cus efflux system. However, here we show that this mutation’s effectiveness is significantly enhanced by epistatic interactions with additional mutations in regulatory genes such as ompR, rho, and fur. These interactions reconfigure global stress response networks, resulting in robust and varied resistance strategies across different populations. This study underscores the critical role of epistasis in bacterial adaptation, illustrating how interactions between multiple mutations and how genetic backgrounds shape the resistance phenotypes of E. coli populations. This work also allowed for refinement of our model describing the role TCRS genes play in bacterial adaptation by now emphasizing that adaptation to environmental stressors is a complex, context-dependent process, driven by the dynamic interplay between genetic and environmental factors. These findings have broader implications for understanding microbial evolution and developing strategies to combat antimicrobial resistance.
more » « less- Award ID(s):
- 1900220
- PAR ID:
- 10549345
- Editor(s):
- Shimada, Tomohiro
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Microorganisms
- Volume:
- 12
- Issue:
- 10
- ISSN:
- 2076-2607
- Page Range / eLocation ID:
- 2000
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Spiers, Andrew (Ed.)more » « less
Introduction: Two-component response systems (TCRS) are the main mechanism by which prokaryotes acclimate to changing environments. These systems are composed of a membrane bound histidine kinase (HK) that senses external signals and a response regulator (RR) that activates transcription of response genes. Despite their known role in acclimation, little is known about the role TCRS play in environmental adaptation. Several experimental evolution studies have shown the acquisition of mutations in TCRS during adaptation, therefore here we set out to characterize the adaptive mechanism resulting from these mutations and evaluate whether single nucleotide changes in one gene could induce variable genotype-by-environment (GxE) interactions.Methods: To do this, we assessed fitness changes and differential gene expression for four adaptive mutations incusS , the gene that encodes the HK CusS, acquired byEscherichia coli during silver adaptation.Results: Fitness assays showed that as the environment changed, each mutant displayed a unique fitness profile with greatest fitness in the original selection environment. RNAseq then indicated that, in ± silver nitrate, each mutant induces a primary response that upregulatescusS, its RRcusR , and constitutively expresses the target response genescusCFBA . This then induces a secondary response via differential expression of genes regulated by the CusR through TCRS crosstalk. Finally, each mutant undergoes fitness tuning through unique tertiary responses that result in gene expression patterns specific for the genotype, the environment and optimized for the original selection conditions.Discussion: This three-step response shows that different mutations in a single gene leads to individualized phenotypes governed by unique GxE interactions that not only contribute to transcriptional divergence but also to phenotypic plasticity. -
more » « less
Abstract Epistasis is caused by genetic interactions among mutations that affect fitness. To characterize properties and potential mechanisms of epistasis, we engineered eight double mutants that combined mutations from the rho and rpoB genes of Escherichia coli. The two genes encode essential functions for transcription, and the mutations in each gene were chosen because they were beneficial for adaptation to thermal stress (42.2 °C). The double mutants exhibited patterns of fitness epistasis that included diminishing returns epistasis at 42.2 °C, stronger diminishing returns between mutations with larger beneficial effects and both negative and positive (sign) epistasis across environments (20.0 °C and 37.0 °C). By assessing gene expression between single and double mutants, we detected hundreds of genes with gene expression epistasis. Previous work postulated that highly connected hub genes in coexpression networks have low epistasis, but we found the opposite: hub genes had high epistasis values in both coexpression and protein–protein interaction networks. We hypothesized that elevated epistasis in hub genes reflected that they were enriched for targets of Rho termination but that was not the case. Altogether, gene expression and coexpression analyses revealed that thermal adaptation occurred in modules, through modulation of ribonucleotide biosynthetic processes and ribosome assembly, the attenuation of expression in genes related to heat shock and stress responses, and with an overall trend toward restoring gene expression toward the unstressed state.
-
The prevalence of multidrug-resistant bacteria and their increased pathogenicity has led to a growing interest in metallic antimicrobial materials and bacteriophages as potential alternatives to conventional antibiotics. This study examines how resistance to excess iron (III) influences the evolution of bacteriophage resistance in the bacterium Escherichia coli. We utilized experimental evolution in E. coli to test the effect of the evolution of phage T7 resistance on populations resistant to excess iron (III) and populations without excess iron resistance. Phage resistance evolved rapidly in both groups. Dual-resistant (iron (III)/phage) populations were compared to their controls (excess iron (III)-resistant, phage-resistant, no resistance to either) for their performance against each stressor, excess iron (III) and phage; and correlated resistances to excess iron (II), gallium (III), silver (I) and conventional antibiotics. Excess iron (III)/phage-resistant populations demonstrated superior 24 h growth compared to all other populations when exposed to increasing concentrations of iron (II, III), gallium (III), ampicillin, and tetracycline. No differences in 24 h growth were shown between excess iron (III)/phage-resistant and excess iron (III)-resistant populations in chloramphenicol, sulfonamide, and silver (I). The genomic analysis identified selective sweeps in the iron (III) resistant (rpoB, rpoC, yegB, yeaG), phage-resistant (clpX →/→ lon, uvaB, yeaG, fliR, gatT, ypjF, waaC, rpoC, pgi, and yjbH) and iron (III)/phage resistant populations (rcsA, hldE, rpoB, and waaC). E. coli selected for resistance to both excess iron (III) and T7 phage showed some evidence of a synergistic effect on various components of fitness. Dual selection resulted in correlated resistances to ionic metals {iron (II), gallium (III), and silver (I)} and several conventional antibiotics. There is a likelihood that this sort of combination antimicrobial treatment may result in bacterial variants with multiple resistances.more » « less
-
Gao, Beile (Ed.)ABSTRACT Escherichia coli can survive for long periods in batch culture in the laboratory, where they experience a stressful and heterogeneous environment. During this incubation, E. coli acquires mutations that are selected in response to this environment, ultimately leading to evolved populations that are better adapted to these complex conditions, which can lead to a better understanding of evolutionary mechanisms. Mutations in regulatory genes often play a role in adapting to heterogeneous environments. To identify such mutations, we examined transcriptional differences during log phase growth in unaged cells compared to those that had been aged for 10 days and regrown. We identified expression changes in genes involved in motility and chemotaxis after adaptation to long-term cultures. We hypothesized that aged populations would also have phenotypic changes in motility and that motility may play a role in survival and adaptation to long-term cultures. While aged populations did show an increase in motility, this increase was not essential for survival in long-term cultures. We identified mutations in the regulatory gene sspA and other genes that may contribute to the observed differences in motility. Taken together, these data provide an overall picture of the role of mutations in regulatory genes for adaptation while underscoring that all changes that occur during evolution in stressful environments are not necessarily adaptive. IMPORTANCE Understanding how bacteria adapt in long-term cultures aids in both better treatment options for bacterial infections and gives insight into the mechanisms involved in bacterial evolution. In the past, it has been difficult to study these organisms in their natural environments. By using experimental evolution in heterogeneous and stressful laboratory conditions, we can more closely mimic natural environments and examine evolutionary mechanisms. One way to observe these mechanisms is to look at transcriptomic and genomic data from cells adapted to these complex conditions. Here, we found that although aged cells increase motility, this increase is not essential for survival in these conditions. These data emphasize that not all changes that occur due to evolutionary processes are adaptive, but these observations could still lead to hypotheses about the causative mutations. The information gained here allow us to make inferences about general mechanisms underlying phenotypic changes due to evolution.more » « less
-
Hendrickson, Heather (Ed.)Abstract Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria–phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.more » « less
