Mutations in the Global Transcription Factor CRP/CAP: Insights from Experimental Evolution and Deep Sequencing

Abstract

The Escherichia coli cyclic AMP receptor protein (CRP or catabolite activator protein, CAP) provides a textbook example of bacterial transcriptional regulation and is one of the best studied transcription factors in biology. For almost five decades a large number of mutants, evolved in vivo or engineered in vitro, have shed light on the molecular structure and mechanism of CRP. Here, we review previous work, providing an overview of studies describing the isolation of CRP mutants. Furthermore, we present new data on deep sequencing of different bacterial populations that have evolved under selective pressure that strongly favors mutations in the crp locus. Our new approach identifies more than 100 new CRP mutations and paves the way for a deeper understanding of this fascinating bacterial master regulator.

Keywords: Carbon catabolite repression; Experimental evolution; Global transcriptional regulation; cAMP receptor protein.

Fig. 1

Network of global transcription factors and sigma factors in Escherichia coli. Illustrated is a) The sigma factors (orange circles) and the 14 biggest regulators by regulon size (grey circles). The size of the circles is directly proportional to the sigmulon or regulon size by number of genes directly affected (RegulonDB 04-03-2019) [19]. Arrows designate regulation of regulator expression; sigma factor transcription (grey arrows), activation (green, arrowheads), repression (red, perpendicular line ends) or dual regulation (blue, reverse arrowheads) [26]. b) The sigma factors (orange circles) and 207 regulators (grey circles) of E. coli plotted by the number of promoters or binding sites recognized, respectively, and the number of genes directly affected (RegulonDB 04-03-2019) [19].

Fig. 2

Illustration of functional domains in a closed and an open conformation of CRP. The locations of functionally important CRP domains in the tertiary structure of apo-CRP (left) and CRP-cAMP₂ (right). The protein structures are from Protein Data Bank entries 3FWE (apo-CRP) and 1ZRC (CRP-cAMP₂) and were modified using the PyMOL Molecular Graphics System, Version 2.3 Schrödinger, LLC.

Fig. 3

Illustration of adaptive mutations identified in CRP a) The locations of functionally important CRP domains in the primary amino acid sequence. b) Locations of mutations identified in the primary and tertiary structure of CRP in previous studies (upper black bar and structure to the left) and in our laboratories by deep sequencing (lower black bar and structure to the right). The number of variants identified per residue is colour coded (black: 0 variants, yellow: 1 variant, orange: 2–3 variants, and red: more than three variants).

Fig. 4

Heatmap of the mutation frequencies (%) of each CRP residue in an E. coli ΔcyaA pTIG strain. Strain and evolution experiment set-up were as previously described [48]. Biomass samples of the populations were taken from preculture (day 0) or from one of 20 identical plates during the evolution experiment (days 1–35). Furthermore, samples were taken at day 7 of the ΔcyaA strain supplemented with 0.1 mM cAMP or 0.5 mM cGMP, and of E. coli ΔcyaA strains with CRP A144T or CRP A144E mutations. The crp gene was sequenced by amplicon next-generation sequencing and the data processed by CLC Genomics Workbench (Qiagen, Aarhus, Denmark).