Global pleiotropic effects in adaptively evolvedEscherichia colilacking CRP reveal molecular mechanisms that define growth physiology

Abstract

Bacterial gene expression is governed by two synergistic mechanismsgrowth-rate dependent global machinery (e.g. ribosomes, RNA polymerase, metabolites and cofactors) and transcription factors that work together for optimal growth. However, in presence of glucose, coordination of global transcriptional regulator cAMP-CRP with such physiological resources, remain elusive.Here, we reveal that the deletion of CRP results in metabolic dysregulation with significant perturbation in protein biosynthesis machinery coupled to impaired glucose import.Additionally, we quantitatively demonstrate that the loss of CRP unbalances proteome allocation, favouring stress or hedging related functions over growth-enhancing functions. To address how Escherichia coli can cope up with such a system-wide upset, we adaptively evolved Δcrp mutant in the presence of glucose. We explicitly show that adaptation to loss of CRP frequently occurs via mutations in the ptsG promoter. This causes higher glucose uptake, resulting in the restoration of the levels of the protein biosynthesis machinery and metabolic rewiring of ATP towards synthesis of costly amino acids along with reduction in unnecessary cAMP synthesis. Thus we elucidate the molecular events underlying growth optimality of the organism, driven by CRP.Here, using a multi-omics (genomics, transcriptomics, metabolomics and phenomics) approach, we illustrate the evolutionary importance of CRP in glucose minimal media conditions. First, we elucidate that evolution redress the loss of CRP by repeatedly accumulating mutations in the intergenic region of the ptsG gene, enabling increased glucose uptake rates and fitness benefit for the organism. Next, we unravel how such adaptation bring about system-wide changes by modulating the global gene expression states and the levels of several key intracellular metabolites that coordinate protein biosynthesis machinery and metabolism. Finally, restoration mechanism as a result of evolution involved fine tuning of proteome allocation in favour of growth and away from stress or hedging functions. Further, using a genome-scale model we were able to quantitatively detail the energetic inefficiencies of the evolved populations which explained their inability to grow optimally as wild-type. Overall, the evolution of Δcrp strain suggests an underlying paradigm which delineates the inherent constraints of genetic and metabolic networks in E. coli.An E. coli K-12 MG1655 (CGSC#6300), was used as the parent strain in this study. We constructed Δcrp knockout in this genetic background by -Red mediated recombination (21), using plasmids pKD46, pKD13 and pCP20. The generation of the knockout strain was confirmed by PCR with custom made oligo-nucleotides against the genomic DNA followed by Sanger sequencing. Generation of Δfis, Δmlc, ΔfisΔcrp and ΔmlcΔcrp strains for growth studies were also generated using the same procedure. Physiological CharacterizationFor transcriptome, metabolome and phenotype characterizations, experiments were performed in 500 mL bioreacto...

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin

“…The absence of CRP exhibited a growth defect in both exponential and stationary phases, as observed in Vibrio vulnificus ( Kim et al, 2013 ), E. coli ( Basak et al, 2014 ), K. pneumoniae ( Ou et al, 2017 ; Lin et al, 2018 ), and Haemophilus parasuis ( Jiang et al, 2020 ). These observations are in accordance with previous reports that explain how growth fitness is affected by the crp deletion, which produces fluctuations of metabolic gene expression and alterations of carbon metabolites, α-ketoacids, cAMP, and amino acids that promote proper coordination of protein biosynthesis machinery during metabolism ( Klumpp et al, 2009 ; Berthoumieux et al, 2013 ; You et al, 2013 ; Pal et al, 2020 ). CRP is a central regulator of carbon metabolism and has been implicated as an important facilitator of host colonization and virulence in many bacterial pathogens, including K. pneumoniae ( Xue et al, 2016 ; Lin et al, 2018 ; Panjaitan et al, 2019 ).…”

Determination and Dissection of DNA-Binding Specificity for the Thermus thermophilus HB8 Transcriptional Regulator TTHB099

“…Notably, the biological roles of the most affected operons in the TTHB099-deficient strain were involved in metabolic pathways that have been reported to be regulated by the archetype CRP Ec [31]. For example, ribosome related genes were downregulated in the absence of TTHB099, similar to what Pal et al reported for their evolutionary expressed CRP Ec -deficient strains [32]. Likewise, iron transport genes were downregulated in the absence of TTHB099, similar to what was observed in the absence of CRP Ec , as Zhang et al reported [33].…”

Global Transcriptional Regulators Fine-Tune the Translational and Metabolic Efficiency for Optimal Growth of Escherichia coli

“…7 ), limiting the potential of the system to attain a growth rate similar to that of the WT ( 12 , 53 – 55 , 57 ). Overall, in the absence of global regulators, we propose that the balance between growth rate and glucose uptake rate is attenuated, making the organism incapable to attain a fitter phenotype, unless forced upon a selection pressure ( 59 , 60 ).…”

“…Mechanistically, this delineated the complementary role of global transcriptional regulators with growth rate-dependent global machinery, predominant in exponentially growing cells. Such analyses can be extended to other global regulators such as cAMP receptor protein (CRP) ( 11 , 59 , 61 ), HNS ( 60 ), Lrp ( 62 ), and Fis ( 63 ), which tempts us to anticipate similar underlying mechanisms of regulation, presumably not confined only to glucose fermentative metabolism.…”