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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 IIA^Glc [Microbiology].

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] and further characterized in 2001 [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 includes 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.  See the Escherichia coli K-12 (strain MG1655) mosaic, from the National Center for Biotechnology Information (NCBI).

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 [Medline].  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').  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 IIA^Glc (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 IIA^Glc, 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 IIA^Glc.  The finding that pla is under CRP-cAMP control was later confirmed by using a mutant of Yersinia pestis lacking CRP [Infect Immun].

Vibrio cholerae adenylate cyclase and Enzyme IIA^Glc 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].
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].  Incidentally in Vibrio fischeri phosphorylated Enzyme IIA^Glc has been unexpectedly implicated in some peculiar functions [J Bacteriol].

Pasteurella multocida adenylate cyclase was shown to be regulated by Escherichia coli Enzyme IIA^Glc [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 [Medline].

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 IIA^Glc or ptsI encoding Enzyme I) lowered the transformation efficiency that could be restored upon addition of cAMP [J Bacteriol].  These findings were later corroborated by the discovery of a novel cAMP receptor-dependent regulon characterized by the presence of a 22bp CRE (Competence Regulatory Element) in promoters of competence-associated genes [J Mol Biol] [J Mol Biol].  This regulon was further characterized in E. coli and other Gammaproteobacteria [Nucleic Acids res].  Interestingly competent genes in E. coli are similarly regulated by CRP-cAMP [J Bacteriol].  Thus questioning the regulation of AC by Enzyme IIA^Glc in H. influenzae is of interest.  Cyclic AMP phosphodiesterase is 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 (probable phosphotransferase protein, 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 [Medline].  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 IIA^Glc specifically 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 IIA^Glc need to be established and defined possibly in E. coli.

For information on pathogenicity of the above-listed bacteria the Bad Bug Book from the U.S. Food and Drug Administration (FDA) is worthy to be visited, particularly for Escherichia coli O157:H7, Vibrio cholerae, Salmonella spp. and Yersinia spp.

To Chapter VII: Purification of adenylate cyclase