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Escherichia coli adenylate cyclase homepage: Chapter VI

Characterization of adenylate cyclase mutants

Three amino-acids of Escherichia coli K12 adenylate cyclase - arginine 188, aspartic acid 414 and glycine 463 - were identified in vivo as essential residues for the activation process by phosphorylated Enzyme IIAGlc [Microbiology].

Essential Residues

Comparison of protein sequences from complete genomes allowed the definition of Clusters of Orthologous Groups of proteins or COGs, as defined by Tatusov, Koonin and Lipman in 1997 [Science Magazine], further characterized in 2001 [Nucleic Acids Res] and further expanded and improved in 2015 [Nucleic Acids Res].  Because 'orthologs' generally have the same function, all members of a COG are assigned a function.  An updated version of the COG database originally included 7 eukaryotic genomes [BMC Bioinformatics].  The COGs have been classified in color-coded functional categories, thus a complete genome can be viewed as a mosaic.

The specific COG3072 assemble 9 adenylate cyclases from Escherichia coli K12, Escherichia coli O157:H7 strain EDL933, Escherichia coli O157:H7 strain Sakai, Yersinia pestis and Salmonella typhimurium, Enterobacteriaceae; Vibrio cholerae, Vibrionaceae; Pasteurella multocida and Haemophilus influenzae, Pasteurellaceae; and Pseudomonas aeruginosa, Pseudomonadaceae.  COG3072 belongs to the 'F' functional category nucleotide transport and metabolism.  COG3072 relates to class I adenylate cyclase as previously defined [PubMed].  With genome sequencing the list of class I adenylate cyclases is slowly increasing in number.

Residue R188 of Escherichia coli K12 adenylate cyclase is present in all COG3072 adenylate cyclases within a conserved stretch of 8 amino-acids, LLLDEFYR.  See the multiple sequence alignment showing conserved amino-acids among COG3072 adenylate cyclases, from NCBI; select 'Color Bits: Identity' before clicking 'Reformat' (note the Identity option may not work).  Residue D414 is conserved in all COG3072 adenylate cyclases.  Residue G463 is conserved in all COG3072 adenylate cyclases except Pseudomonas aeruginosa where G is replaced with Q (G and Q are both polar and neutral but G is 'neutral and small' and Q 'polar and relatively small').

Other conserved amino-acids of E. coli catalytic domain (S103, S113, D114, D116, W118, E185, W200, K260, K264, D300 and K332) were found to be essential for activity in vitro [Biochem J].  The non-conserved residue T189 was also found to be essential however R188 was not examined.  Other conserved residues were found nonessential (R19, S106, K136, E242, W249, K253, W374, Y394).

Bacteria that possess an E. coli-like adenylate cyclase also possess an E. coli-like Enzyme IIAGlc (even though it may not be used for glucose transport).  The specific COG2190 assembles Enzyme IIA components of the phosphotransferase system (PTS).  COG2190 belongs to the 'G' functional category carbohydrate transport and metabolism.

To date, protein sequence comparisons seem to emphasize the essentiality of the three amino-acids mentioned above.  Do they also infer -to a certain extent- conservation of the regulatory mechanism, i.e., the activation by phosphorylated Enzyme IIAGlc, among the present group of adenylate cyclases?  An answer to this question is ambiguous however one can substantiate a positive answer by looking at experimental data.

In Yersinia pestis the CRP-cAMP complex controls the transcription of pla, the plasminogen activator gene [J Bacteriol].  In the presence of glucose, transcription of pla was reduced indicating that Y. pestis adenylate cyclase is possibly regulated by phosphorylated Enzyme IIAGlc.  The finding that pla is under CRP-cAMP control was later confirmed by using a mutant of Yersinia pestis lacking CRP [Infect Immun].  Furthermore the unique posttranscriptional regulation of Y. pestis CRP is required for the transcription of pla [MBio].

Vibrio cholerae adenylate cyclase and Enzyme IIAGlc were both identified by sequence similarity.  Studying the regulation of V. cholerae adenylate cyclase may be of interest physiologically as it was demonstrated that cAMP via its receptor protein is involved in expression of the cholera toxin [Proc Natl Acad Sci U S A].  Also, synthesis of a metalloprotease involved in pathogenicity is dependent on the cAMP receptor protein during entry in stationary phase [J Bacteriol].  Transcription of genes involved in biofilm polysaccharide and protein synthesis is negatively controlled by the CRP-cAMP complex, either directly or indirectly [J Bacteriol].  Additionally, phosphorylated Enzyme I has been proposed to be involved in regulation of surface-associated growth as well as exopolysaccharide synthesis [J Bacteriol], and the PTS has been implicated in colonization of the germ-free mouse intestine [Infect Immun].  In particular, expression of the mtl operon encoding the mannitol-specific PTS of V. cholerae is dependent on the presence of a small regulatory RNA (MtlS sRNA) [J Bacteriol].  It was proposed the PTS amino sugar GlcNAc is used by V. cholerae during colonization of the host intestine [PLoS One]; see also [PubMed Commons].  The proposed role of the PTS, particularly dephosphorylated Enzyme IIAGlc, in V. cholerae chitin transport and metablism [J Bacteriol] should be taken with caution [PubMed Commons].  Noteworthy, cyaA or crp mutant strains of V. cholerae could be used as indicators for monitoring the presence of V. cholerae phages in the water [Appl Environ Microbiol].

In Vibrio fischeri, reclassified Aliivibrio fischeri in 2007 [Int J Syst Evol Microbiol], phosphorylated Enzyme IIAGlc has been unexpectedly implicated in some peculiar functions [J Bacteriol].  In A. fischeri ES114, bioluminescence was found to be regulated by CRP-cAMP [J Bacteriol].  In Vibrio vulnificus, the TonB3 system is CRP-dependent [J Bacteriol].

Pasteurella multocida adenylate cyclase was shown to be regulated by Escherichia coli Enzyme IIAGlc [J Bacteriol]. The regulatory process may therefore be conserved in P. multocida.  Incidentally, the cya gene of P. multocida was detected in all tested subspecies and serogroups of P. multocida [PubMed].

In Haemophilus influenzae, cAMP is essential to competence for DNA transformation [J Bacteriol].  It was shown that mutations in PTS genes (either crr encoding Enzyme IIAGlc or ptsI encoding Enzyme I) lowered the transformation efficiency that could be restored upon addition of cAMP [J Bacteriol].  Thus questioning the regulation of AC by Enzyme IIAGlc in H. influenzae is of interest.  Later on, a novel cAMP receptor-dependent regulon characterized by the presence of a 22bp CRE (Competence Regulatory Element) in promoters of competence-associated genes was discovered [J Mol Biol] and further characterized [J Mol Biol].  This regulon was studied in E. coli and other Gammaproteobacteria [Nucleic Acids res].  Interestingly competent genes in E. coli were also regulated by CRP-cAMP [J Bacteriol] however physiological conditions leading to competence in E. coli have not yet been defined [Mol Microbiol].  Cyclic AMP phosphodiesterase was also involved in regulating the cAMP level [J Bacteriol].

Experimental data are not presently available for Pseudomonas aeruginosa as regards the regulation of adenylate cyclase.  Visit the Pseudomonas Genome Database and search the database for PA5272 (adenylate cyclase, cyaA, COG3072) and PA3760 (N-Acetyl-D-Glucosamine phosphotransferase system transporter, COG2190).  P. aeruginosa virulence is not significantly attenuated in a mutant strain lacking cyaA [Infect Immun].  In fact P. aeruginosa membrane-bound adenylate cyclase (encoded by cyaB) is more likely to play a prominent role in virulence [PubMed].  Unlike E. coli, cAMP levels in P. aeruginosa do not vary significantly with the carbon source used in the culture medium [J Bacteriol].  Also, even though the cAMP receptor of P. aeruginosa (Vfr) is a global regulator of transcription [Microbiology], it does not play a role in catabolite repression [Microbiology] as previously inferred [J Bacteriol].  Actually a small RNA (CrcZ) has been described as global regulator of catabolite repression [Proc Natl Acad Sci U S A].  These results however do not rule out a regulatory mechanism for P. aeruginosa adenylate cyclase possibly involving the so-called 'probable phosphotransferase protein'.

Presently, it is difficult to draw further conclusion as regards the regulation of adenylate cyclase by phosphorylated Enzyme IIAGlc in bacteria possessing an E. coli-like adenylate cyclase.  However it is legitimate, considering the data reported above, to question the extent of such regulation in any of these affiliated bacteria.  Nonetheless, in order to be able to analyze sequencing data with greater insight the putative molecular interactions between adenylate cyclase and phosphorylated Enzyme IIAGlc need to be established and defined possibly in E. coli.

The Bad Bug Book from the U.S. Food and Drug Administration (FDA) gives information (and references to the Centers for Disease Control and Prevention) on pathogenicity of the above-listed bacteria, particularly Escherichia coli O157:H7, Vibrio cholerae, Salmonella spp. and Yersinia spp.

Finally, it is to be mentioned the role of cAMP in the different phases of a pathogenic process is difficult to assess.  Generally a glucose effect on motility, and/or expression of pathogenic factors, and/or bacterial communication (quorum sensing) and/or biofilm formation, suggests a role for cAMP in host-pathogen interactions.  However, studies using cya and crp mutant strains have to be taken with caution because these mutants suffer many defects, particularly they grow very slowly on a limited number of carbon sources.  Also, levels of cAMP most likely vary during the infection process therefore new approaches need to be determined to analyze the effect of cAMP at different phases of the infection, for example upon exhaustion of a glucose-like carbon source [PubMed Commons].  A strategy was devised to maintain relatively high and constant levels of cAMP in V. cholerae by deleting all IIC domains of the PTS.  However, because PTS transport was fully eliminated, regulations by PTS transport, including regulation of cAMP levels and inducer exclusion, were also eliminated [Mol Microbiol] [PubMed Commons].

To Chapter VII: Purification of adenylate cyclase   Chapter VII