Introduction to CRP, the Cyclic AMP Receptor Protein of Escherichia coli, physiological effects
Adenylate cyclase catalyzes the transformation of adenosine 5'-triphosphate (ATP) into cyclic adenosine 3’, 5’-monophosphate (cAMP), CYAA at UniProtKB. In Escherichia coli and related bacteria, cAMP controls gene expression (Botsford JL, 1992) . This control occurs via the Catabolite gene Activator Protein (CAP, Zubay G, 1970), also called Cyclic AMP Receptor Protein (CRP, Emmer M, 1970), CRP at UniProtKB. In E. coli, The CAP family of proteins includes FNR and YeiL.
CRP exists as a homodimer, each subunit is composed of two domains connected by a small hinge, the N-terminal cAMP-binding domain and the C-terminal DNA-binding domain, CRP at RCSB PDB. Upon binding of cAMP, the long helices at the dimer interface reposition leading to activation of the CRP-cAMP complex (Youn H, 2006). The mechanism by which CRP-cAMP activates transcription was the object of an interesting review by Tutar Y, 2008. The much-awaited structure of CRP without cAMP was reported by Popovych N, 2009.
When bound to cAMP, CRP binds to specific sites upstream of promoters, causing transcriptional activation or repression (Kolb A, 1993). Interestingly, it has been inferred that CRP optimally 'responds' to physiological cAMP concentrations consequently hindering activation by ligands other than cAMP (Youn H, 2008).
CAP-dependent activation of transcription upon binding of cAMP
The mechanisms of CRP-dependent activation of transcription at class I and II promoters have been described by Busby S, 1999, and further characterized (Lawson CL, 2004; Miroslavova NS, 2006; Hollands K, 2007). The CRP-cAMP complex can act as a transcriptional regulator by competing for DNA binding or forming complexes with other transcriptional regulators, for example CytR (Gavigan SA, 1999; Kallipolitis BH, 2004). It can also act as a roadblock for RNA polymerase at a class I promoter (Jaeger T, 2008). Transcriptional repression may result from preventing activation by CRP-cAMP (Wickstrum JR, 2010). It was suggested that upstream-bound CRP-cAMP can repress transcription by restraining RNA polymerase (Lee DJ, 2012). Nakano M, 2014 studied promoters with two CRP-binding sites, particularly using the pck gene encoding phosphoenolpyruvate carboxykinase as a model promoter (Comment). Also, CRP can bind to thousands of different sites without affecting transcription initiation of genes located within these sites (Grainger DC, 2005), and it was proposed that CRP may act as a nucleoid-associated protein involved in the organization of the chromosome (Visweswariah SS, 2015, Comment).
The CRP-cAMP complex is a positive transcriptional regulator of a number of catabolite operons (Zubay G, 1970), and as such plays a role in catabolite repression (Magasanik B, 1961). Catabolite repression, also known as carbon catabolite repression, was originally defined as the mechanism by which glucose or another rapidly metabolized carbohydrate exerts a continued inhibition on synthesis of catabolic enzymes (Crasnier M, 1996). Catabolite repression has been extensively studied in enteric bacteria, particularly the lactose operon (Silverstone AE, 1972). The role of cAMP as an antagonist of catabolite repression is well established (Ullmann A, 1968). Cyclic AMP-independent mutants of CRP have been isolated that relieve catabolite repression (Karimova G, 2004), and attempts to release catabolite repression are undertaken to genetically engineer microbial biocatalysts (Sievert C, 2017, Comment).
Over the years, catabolite repression has gained a broader definition, and usualy refers to the mechanism by which glucose exerts inhibition on the transcription of any gene. Analysis of 'CRP-dependent glucose repression' revealed that transcription of specific genes may be enhanced by CRP-cAMP (Gosset G, 2004). Novel predicted binding sites were identified in E. coli that may be functional for CRP (Brown CT, 2004), and unknown CRP regulons have been revealed by using a run-off transcription/microarray analysis (ROMA; Zheng D, 2004). Groups of genes displaying a transcriptional response under condition of glucose repression were further defined using transcriptome data (Gutierrez-Ríos RM, 2007). Also, mathematical modeling has been applied for describing the effects of variable concentrations of CRP-cAMP, and models were compared for their predictive value (Kremling A, 2009). A 'fine-tuning' of transcriptional regulation by CRP-cAMP, via the level of cAMP, was described involving the base-pairing RNA Spot 42 (Beisel CL, 2011) however, data analysis indicates participation of Spot 42 may have been overstated. Incidentally, Spot 42 sRNA was reported to affect L-arabinose uptake under specific conditions (Chen J, 2016).
As it happens, the CRP-cAMP complex regulates transcription of many genes, and therefore by controlling gene expression, cAMP is involved in many aspects of E. coli physiology. Of importance, early reports indicated that CRP-cAMP may regulate the transcription of genes involved in cell division. However, it was later established that CRP-cAMP is not essential for cell division (D'Ari R, 1988). Also, even though it was reported that CRP-cAMP regulates the synthesis of enzymes belonging to the tricarboxylic acid (TCA) cycle, the TCA cycle flux does not seem to be affected by the absence of the CRP-cAMP complex (Perrenoud A, 2005). The effect of CRP-cAMP on the TCA cycle flux was only observed under certain specific conditions (Nanchen A, 2008).
Cyclic AMP however is not essential for survival, as E. coli cyaA strains lacking adenylate cyclase are viable (Brickman E, 1973). However, because of the transcriptional regulation by CRP-cAMP, cyaA and crp strains (lacking CRP) present some unique features. For example, they do not grow on a large variety of carbon sources including lactose, maltose, arabinose, mannitol and glycerol (Perlman RL, 1969), are not motile and therefore incapable of chemotactic responses, are resistant to antibiotics including fosfomycin (Nilsson AI, 2003), fosmidomycin (Sakamoto Y, 2003), nalidixic acid, streptomycin and mecillinam (Aono R, 1979), are pH-sensitive for growth (Ahmad D, 1988) and better hosts for recombinant expression systems, especially in stationary phase (Grossman TH, 1998). The proposal cya mutant strains have a higher level of persistance must be taken with caution (Molina-Quiroz RC, 2018, Comment). Interestingly, investigation of the effect of a crp deletion indicated that epistasis is a crucial factor in adaptive evolution of E. coli laboratory lines (Cooper TF, 2008).
Transcription of the cyaA gene is negatively regulated by CRP-cAMP (Mori K, 1985), as well as transcription of the crp gene (Aiba H, 1983). However, in both cases the negative transcriptional control by CRP-cAMP does not seem to cause major effects in vivo, at least under current experimental conditions (Kolb A, 1993). In particular, it was shown by using gene fusion techniques that neither cAMP nor CRP-cAMP plays a major role in transcriptional or translational regulation of cyaA expression (Bankaitis VA, 1982). In Salmonella typhimurium, transcription of cyaA is also repressed by CRP-cAMP (Fandl JP, 1990). Translation of adenylate cyclase mRNA is ineffective (Roy A, 1988), as to prevent excessive synthesis of adenylate cyclase. This can be attributed to the fact that excess cAMP is toxic to E. coli.
Transcription of crp is additionally regulated by the DNA-binding protein FIS, with both FIS and CRP-cAMP being required for repression of crp transcription (González‐Gil G, 1998), FIS at RCSB PDB. Incidentally, the study of FIS has led to the concept of a transcription factor acting as a local 'topological homeostat' (Muskhelishvili G, 2003). Additionally, in vitro studies have indicated that FIS may play a glucose-dependent repressive role at the nrf promoter thus being CRP-like (Browning DF, 2005). A physiological role for FIS and CRP together was also reported particularly in a rich growth medium (Galán B, 2008). Transcription of a plasmid-encoded toxin gene from pathogenic E. coli is also regulated by both FIS and CRP (Rossiter AE, 2010). Finally, crp expression was found not to be regulated post-transcriptionally by Hfq-dependent sRNAs (Lee HJ, 2016). However, an effect of sRNA SdsR was reported in late stationary phase (Fröhlich KS, 2016, Comment).
Because intracellular cAMP concentrations vary with the carbon source used for bacterial growth, any gene whose transcription is regulated by CRP-cAMP may have various levels of expression depending on growth conditions, especially the available carbon source. Correspondingly, the CRP-cAMP complex has been implicated in the establishment of foraging-like behavior when the carbon source is unfavorable (Liu M, 2005; Zhao K, 2007).
