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Introduction: Cyclic AMP in Escherichia coli

Adenylate cyclase (AC) is the enzyme that catalyzes the transformation of adenosine 5'-triphosphate (ATP) into cyclic adenosine 3’, 5’-monophosphate (cAMP) AC at UniProtKB.

In Escherichia coli and related bacteria, cAMP controls gene expression [Microbiol Mol Biol Rev].  This control occurs via the Catabolite gene Activator Protein (CAP) [Proc Natl Acad Sci U S A] also called Cyclic AMP Receptor Protein (CRP) [Proc Natl Acad Sci U S A].  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 UniProtKB CRP at RCSB PDB.  Upon binding of cAMP, the long helices at the dimer interface reposition leading to activation of the CRP-cAMP complex [J Biol Chem].  The mechanism by which CRP-cAMP activates transcription initiation is still under scrutiny [Cell Biochem Funct].  The much-awaited structure of CRP without cAMP was reported in 2009 [Proc Natl Acad Sci U S A]; see Kalodimos Group Home Page for a movie.

When bound to cAMP, CRP binds to specific sites upstream of promoters, causing transcriptional activation or repression [Annu Rev Biochem].  Interestingly it has been inferred that CRP optimally 'responds' to physiological cAMP concentrations consequently hindering activation by ligands other than cAMP [J Bacteriol].

CAP-dependent activation of transcription upon binding of cAMP
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 and Ebright [PubMed] and further characterized [PubMed] [Biochem Soc Trans] [FEMS Microbiol Lett].  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 [J Biol Chem] [PubMed].  It can also act as a roadblock for RNA polymerase at a class I promoter [J Bacteriol].  Transcriptional repression may result from preventing activation by CRP-cAMP [J Bacteriol].  Also it was suggested that upstream-bound CRP-cAMP can repress transcription by restraining RNA polymerase [mBio].

The CAP family of proteins

The CRP-cAMP complex is a positive transcriptional regulator of a number of catabolite operons [Proc Natl Acad Sci U S A] and as such plays a role in catabolite repression [PubMed].  Catabolite repression, also known as carbon catabolite repression, is the mechanism by which glucose or another rapidly metabolized carbohydrate exerts a continued inhibition on synthesis of catabolic enzymes [Res Microbiol].  Catabolite repression has been extensively studied in enteric bacteria, particularly in the case of the lactose operon [J Bacteriol].  The role of cAMP as an antagonist of catabolite repression [PubMed] is well established [PubMed].  Cyclic AMP-independent mutants of CRP have been isolated that relieve catabolite repression [PubMed].

When catabolite repression occurs, a transcriptome analysis revealed that transcription of specific genes may be enhanced by CRP-cAMP [J Bacteriol].  Groups of genes displaying a transcriptional response under condition of catabolite repression were further defined using transcriptome data [BMC Microbiol].  Also mathematical modeling has been applied to catabolite repression and models were compared for their predictive value [FEBS J].

In 2011, a 'fine-tuning' of transcriptional regulation by CRP-cAMP, via the level of cAMP, was described involving the base-pairing RNA Spot 42 [Mol Cell].  However, based on data presented the participation of Spot 42 in catabolite repression may have been overstated.  Incidentally, Spot 42 sRNA was reported to affect L-arabinose uptake under specific conditions [J Bacteriol].

In addition to its role in regulating transcription of catabolite operons, the CRP-cAMP complex also regulates transcription of many other genes including genes involved in flagella formation [J Bacteriol], biofilm formation [J Bacteriol], nitrogen assimilation [Mol Microbiol] [Nucleic Acids Res], anaerobic growth under nitrate-limited conditions [J Bacteriol], ammonia assimilation [BMC Microbiol], energy metabolism [J Bacteriol], nucleotide biosynthesis [J Bacteriol], iron uptake [FEBS J], membrane functions [J Bacteriol], transport system [J Bacteriol], porin synthesis [Mol Microbiol], outer membrane passage [J Bacteriol], bacterial conjugation [J Bacteriol], osmoprotection [J Bacteriol], stress response [Microbiol Mol Biol Rev], degradation of aromatic compound [Microbiology], small protein synthesis [J Bacteriol], acid resistance [J Bacteriol], stringent response [Cell], cell-to-cell communication [J Bacteriol] [J Bacteriol] [Cell Res], multidrug resistance [J Bacteriol] [Journal Antibiot] [Antimicrob Agents Chemother], cold adaptation [J Bacteriol] and genes encoding enzymes of pharmaceutical importance; for example pga [J Biol Chem].  Furthermore some of the genes that are induced under starvation conditions are subject to transcriptional control by CRP-cAMP [PubMed].  In uropathogenic E. coli, CRP-cAMP regulates the formation of pili associated with pyelonephritis (Pap pili) [Mol Microbiol] as well as type 1 fimbriae [PLoS Pathog] [Environ Microbiol].  In pathogenic Salmonella CRP-cAMP regulates genes related to biofilm formation [Microbiology].

Early reports have 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 [J Bacteriol].

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 [J Bacteriol].  The effect of CRP-cAMP on the TCA cycle flux was however observed under certain specific conditions [J Bacteriol].

Finally, novel predicted binding sites have being identified in Escherichia coli that may be functional for CRP [Proc Natl Acad Sci U S A].  Unknown CRP regulons have also been revealed by using a run-off transcription/microarray analysis (ROMA) [Nucleic Acids Res].  Interestingly, CRP can bind to thousands of different sites without affecting transcription initiation of genes located within these sites [Proc Natl Acad Sci U S A].  Investigation of the effect of a crp deletion indicated that epistasis is a crucial factor in adaptive evolution of Escherichia coli laboratory lines [PLoS Genet].

Escherichia coli cyaA strains lacking adenylate cyclase are viable indicating that cAMP is not essential for survival [J Bacteriol].  However, because of the transcriptional regulation by CRP-cAMP, cyaA strains (or 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 [PubMed], they are not motile and thereby are incapable of chemotactic responses, they are resistant to antibiotics including fosfomycin [Antimicrob Agents Chemother], fosmidomycin [PubMed], nalidixic acid, streptomycin and mecillinam [J Bacteriol], they are pH-sensitive for growth [J Bacteriol], and they are better hosts for recombinant expression systems, especially in stationary phase [PubMed].

Therefore, by controlling gene expression, cAMP is involved in many aspects of Escherichia coli physiology.

Transcription of the cyaA gene is negatively regulated by CRP-cAMP [J Biol Chem], as well as transcription of the crp gene [PubMed].  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 [PubMed].  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 [J Bacteriol].  In Salmonella typhimurium, transcription of cyaA is also repressed by CRP-cAMP [Genetics].

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 [EMBO J] [FIS at RCSB PDB].  Incidentally, the study of FIS has led to the concept of a transcription factor acting as a local 'topological homeostat' [Front Biosci].  Additionally, in vitro studies have indicated that FIS may play a role in catabolite repression [Mol Microbiol].  A physiological role for FIS in catabolite repression was also reported particularly in a rich growth medium [Microbiology].  Interestingly, transcription of a plasmid-encoded toxin gene from pathogenic E. coli was reported to be regulated by both FIS and CRP [Mol Microbiol].  Finally, crp expression was found not to be regulated post-transcriptionally by Hfq-dependent sRNAs [Nucleic Acids Res].  However, an effect of sRNA SdsR was reported in late stationary phase [Nucleic Acids Res] [PubMed Commons].

The translation of adenylate cyclase mRNA is ineffective [PubMed] as to prevent excessive synthesis of adenylate cyclase.  This can be attributed to the fact that overproduction of cAMP is lethal to Escherichia coli possibly due to an accumulation of methylglyoxal [J Bacteriol] [Microbiology].

Translation of adenylate cyclase mRNA

Finally, intracellular cAMP concentrations vary with the carbon source used for bacterial growth.  Therefore, any gene whose transcription is regulated by CRP-cAMP may have various levels of expression depending on growth conditions, especially the carbon source available to the bacteria.  Correspondingly, the CRP-cAMP complex has been implicated in the establishment of foraging-like behavior when the carbon source is unfavorable [J Biol Chem] [Nucleic Acids Res].

Chapter I