Genotype-by-environment interactions govern fitness changes associated with adaptive mutations in two-component response systems

Abstract

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 in cusS, the gene that encodes the HK CusS, acquired by Escherichia 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 upregulates cusS, its RR cusR, and constitutively expresses the target response genes cusCFBA. 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.

Keywords: Escherichia coli; adaptation; fitness; gene-by-environment (GxE) interaction; two-component response systems.

FIGURE 1

Linearized model of the CusS protein and location of adaptive mutations. The CusS monomer can be divided into 5 major subdomains. First, the N-terminal cytoplasmic tail which is composed of the first 15 residues and houses three silver adaptive mutations (L12R, T14P and R15L), two of which are represented in this study. The second domain is the first transmembrane segment (TM1); to date, only the T17P adaptive mutation has been identified in this domain in an EE study. This then leads into the periplasmic sensor domain; there has yet to be adaptive mutations identified in this domain due to EE studies in silver nitrate, although the Long-Term Evolution Experiment (LTEE) did identify one adaptive mutation in this domain (F110L (Barrick and Lenski, 2013; Lenski, 2018)). Next is the second transmembrane domain, which to our knowledge has never acquired any adaptive mutations. Finally, we have the C-terminal kinase domains (which also houses a HAMP domain and a dimerization domain). There have been two adaptive mutations identified in this domain (D435A in the dimerization domain and N279H in the ATP binding pocket) although only N279H is represented in this study. We will note that, we attempted to generate all the mutants above, albeit were only successful at the ones in bold (T14P, R15L, T17P and N279H).

FIGURE 2

Single amino acid changes in one gene can affect organismal fitness which is further influenced by media type and silver nitrate concentrations. Changes in fitness relative to the WT in all four cusS mutants was evaluated in (A) DMB (the original selective media), in varying concentrations of silver nitrate, including the original selection concentration of 50 ng/mL, and in (B) LB at varying concentrations of silver nitrate. The black dotted line represents the fitness of the WT in DMB alone and the black solid line represents the fitness of the WT in LB alone. Here we show that the composition of the media has a clear effect on fitness, and interestingly, mutants show no change in fitness relative to the WT in DMB alone while all show reduced fitness as compared to the WT in LB. In addition, the overall tolerance for the selective agent, silver nitrate, is significantly higher in LB than it is in DMB. Together this data shows that mutant displays a unique fitness pattern and that is the result of each individual genotype interacting to varying degrees with the silver nitrate in its environment. p-values from one-way ANOVAs are given in Table 2.

FIGURE 3

Media state influences fitness. We then evaluated if the state of the media (solid vs. liquid) had an influence on fitness relative to the WT. (A) shows changes in fitness on DMB agar and (B) on LB agar, again both with increasing concentrations of silver nitrate. The black dotted line represents the fitness of the WT in DMB in absence of silver nitrate. The black solid line represents the fitness of the WT in LB in presence of silver nitrate. Overall, the data shows that on agar as with broth, the individual genotypes interact to varying extents on the solid media (DMB vs. LB agar) and the varying concentration of silver nitrate. p-values from one-way ANOVAs are given in Table 3.

FIGURE 4

Individual cusS mutations lead to varying levels of both TCRS and efflux pump expression in absence of silver nitrate. Here we used RNAseq to evaluate changes in expression of both the cusS/R TCRS and the cusCFBA efflux pump genes. (A–D) Shows TCRS gene expression in DMB and LB in absence and presence of silver nitrate. (E–L) Shows expression of the individual efflux pump genes in DMB and LB, again in absence and presence of silver nitrate. The data shows that single point mutations in the cusS gene result in increased expression of the TCRS and constitutive expression of the efflux pump genes. In addition, the patterns of expression for these genes differ with both the individual genotype and the media type used.

FIGURE 5

Single mutations in cusS are pleiotropic and lead to varying cellular responses. Pathway analysis was performed using limma’s (Ritchie et al., 2015) “kegg” functionality. The genes that were considered Up/Down in this analysis were at FDR <.05. Here we show up/down regulated pathways for each mutant in (A) DMB, (B) DMB, with silver nitrate, (C) LB and (D) LB with silver nitrate for the WT and each mutant in each media type supplemented with silver nitrate. X-axis represents the number of genes that have been identified as differentially regulated in that pathway. Data shows that there are overlapping biological processes between all populations, while each mutant shows varying pleiotropic effects through differentially regulating a unique subset of secondary pathways that changes with the genotype, media type and the addition of silver nitrate.

FIGURE 6

Three-step model depicting the adaptive response of cusS silver resistant mutants. Here we propose a 3-step adaptive mechanism associated with adaptation in TCRS genes. This begins with a primary response where mutations in a HK result in increased TCRS expression and constitutive expression in response genes. In this case that is an increased expression of cusS/R and constitutive expression of cusCFBA although expression levels do vary by mutant due to varying GxE interactions. This primary response then controls a secondary response that leads to crosstalk between the RR and non-cognate pathways. Here we see some pathways that are upregulated by all mutants in both types of media, while other pathways are only upregulated by select mutants in select media. Both the primary and secondary responses will come at a fitness cost therefore we propose that fitness is then refined by unique gene expression patterns associated with the individual GxE interactions deemed the tertiary response. We propose that this mechanism may be maintained by all TCRSs that acquire adaptive mutations provided evidence to support the role that TCRS play in environmental adaptation. Figure was created with BioRender.com.