Global pleiotropic effects in adaptively evolved Escherichia coli lacking CRP reveal molecular mechanisms that define the growth physiology.

Ankita Pal,Aswin Sai Narain Seshasayee,Sumana Srinivasan,Mahesh S Iyer,K V Venkatesh

doi:10.1098/rsob.210206

Abstract

Evolution facilitates emergence of fitter phenotypes by efficient allocation of cellular resources in conjunction with beneficial mutations. However, system-wide pleiotropic effects that redress the perturbations to the apex node of the transcriptional regulatory networks remain unclear. Here, we elucidate that absence of global transcriptional regulator CRP in Escherichia coli results in alterations in key metabolic pathways under glucose respiratory conditions, favouring stress- or hedging-related functions over growth-enhancing functions. Further, we disentangle the growth-mediated effects from the CRP regulation-specific effects on these metabolic pathways. We quantitatively illustrate that the loss of CRP perturbs proteome efficiency, as evident from metabolic as well as ribosomal proteome fractions, that corroborated with intracellular metabolite profiles. To address how E. coli copes with such systemic defect, we evolved Δcrp mutant in the presence of glucose. Besides acquiring mutations in the promoter of glucose transporter ptsG, the evolved populations recovered the metabolic pathways to their pre-perturbed state coupled with metabolite re-adjustments, which altogether enabled increased growth. By contrast to Δcrp mutant, the evolved strains remodelled their proteome efficiency towards biomass synthesis, albeit at the expense of carbon efficiency. Overall, we comprehensively illustrate the genetic and metabolic basis of pleiotropic effects, fundamental for understanding the growth physiology.

Highlights

Global transcriptional factors represent a cornerstone in the transcriptional regulatory network (TRN), which facilitates system-wide changes in gene expression levels in response to alterations in its external or internal environment [1,2,3]
The transcriptome of this strain, when compared to its parent wild-type (WT) strain, showed ∼725 differentially expressed (DE) genes (absolute fold change ≥ 2, adjusted p-value less than 0.05) of which ∼534 genes (74%) were downregulated and ∼191 genes (26%) were upregulated in the mutant, indicating a large upset of the global transcriptome. This reiterated the role of CRP as a transcriptional activator, which was in good agreement with previous gene expression studies [9,11,18]
Out of the total 725 DE genes, 346 genes (242 downregulated and 104 upregulated genes) were enriched for KEGG pathways. Among these enriched pathways, downregulated genes were significantly associated with transporters, tricarboxylic acid (TCA) cycle needed for energy generation during aerobic respiration, and carbohydrate metabolism involved in the processing of secondary carbon compounds such as uronic acid, galactitol and glucan metabolism

Summary

Introduction

Global transcriptional factors represent a cornerstone in the transcriptional regulatory network (TRN), which facilitates system-wide changes in gene expression levels in response to alterations in its external or internal environment [1,2,3]. Considering the complex interactions existing within the TRN of an organism, the absence of global transcription factors results in direct or indirect cellular responses that incapacitate the ability to attain favourable phenotypic outcomes, even for a simple prokaryote like Escherichia coli. CRP, along with its cognate signalling molecule cAMP [4,5,6], activates transcription at more than 200 promoters, as evidenced from the genome-wide binding and reporter-based studies in E. coli [7,8,9]. The physiological significance of its activator molecule cAMP, in coordinating the carbon and nitrogen demands via carbon catabolites, addressed the long-standing debate on carbon catabolite repression [31]

Methods

Results

Conclusion