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 (Crasnier M, 1990).
Comparison of protein sequences from complete genomes allowed the definition of 'Clusters of Orthologous Groups of proteins' or COGs, as defined by Tatusov RL, 1997. COGs were further characterized (Tatusov RL, 2001), and further expanded and improved (Galperin MY, 2015). An updated version of the COG database originally included 7 eukaryotic genomes (Tatusov RL, 2003). Because 'orthologs' generally have the same function, all members of a COG are assigned a function. The COGs have been classified in color-coded functional categories, thus a complete genome can be viewed as a mosaic.
The specific COG3072 originally assembled 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', and relates to class I adenylate cyclases as previously defined (Bârzu O, 1994). 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 from NCBI showing conserved amino-acids among COG3072 adenylate cyclases; go to COG3072 and 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'). Thus sequence identities confirm an important role for the three amino-acids previously characterized by Crasnier M, 1990.
Other conserved amino-acids in E. coli catalytic domain (S103, S113, D114, D116, W118, E185, W200, K260, K264, D300 and K332) were found by mutagenesis to be essential for activity in vitro (Linder JU, 2008). 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 may substantiate a positive answer by looking at experimental data.
In Yersinia pestis, the CRP-cAMP complex controls positively the transcription of pla encoding Pla, the plasminogen activator protease (Kim TJ, 2007). In the presence of glucose, transcription of pla was reduced indicating that Y. pestis adenylate cyclase is most likely regulated by phosphorylated Enzyme IIAGlc. The finding that pla is under CRP-cAMP control was later confirmed by using a mutant strain of Yersinia pestis lacking CRP (Zhan L, 2008). Furthermore, the unique posttranscriptional regulation of Y. pestis CRP is required for the transcription of pla (Lathem WW, 2014).
Vibrio cholerae adenylate cyclase and Enzyme IIAGlc were both identified by sequence similarity. Studying the regulation of V. cholerae adenylate cyclase is of particular interest, as it was demonstrated that cAMP via its receptor protein is involved in expression of the cholera toxin (Skorupski K, 1997). Interestingly, transcription of hpaA (encoding a metalloprotease involved in pathogenicity) is dependent on the cAMP receptor protein, occurs upon entry into stationary phase, and is inhibited by the presence of glucose (Silva AJ, 2004). Thus, it appears there is an E. coli-like correlation between glucose transport and cAMP levels. Also, cyaA and crp mutant strains exhibit increased biofilm-forming capacities, and an increase in biofilm formation in wild-type V. cholerae is observed in the presence of glucose (Fong JC, 2008). Furthermore, the PTS was implicated in vurulence gene expression (Wang Q, 2015, Comment). A novel role for phosphorylated Enzyme I was proposed for regulating surface-associated growth as well as exopolysaccharide synthesis (Houot L, 2008). Unlike E. coli, glucose utilization does not necessarily require Enzyme I or HPr however it does require Enzyme IIAGlc (Houot L, 2010). Expression of the mtl operon encoding the mannitol-specific PTS of V. cholerae is uniquely dependent on the presence of a small regulatory RNA (Mustachio LM, 2012). Also, the PTS amino sugar GlcNAc was found to be essential for V. cholerae colonization of the host intestine (Mondal M, 2014, reinforced by Chourashi R, 2016, Comment), a proposal in opposition with the report the PTS has a limited role in infection (Hayes CA, 2017b, Comment). The proposed role of the PTS, particularly dephosphorylated Enzyme IIAGlc, in V. cholerae chitin transport and metabolism should be taken with caution (Yamamoto S, 2017, Comment). Chitin colonization and degradation are unlikely to be under catabolite repression in V. cholerae natural habitat (Blokesch M, 2012, Comment). Chitin utilization was analyzed by genetic 'dissection' (Hayes CA, 2017a, Comment). Noteworthy, cyaA or crp mutant strains of V. cholerae are susceptible to certain phages, and thus could be used for monitoring V. cholerae phages (Zahid MS, 2010).
In Vibrio fischeri, reclassified as Aliivibrio fischeri (Urbanczyk H, 2007), phosphorylated Enzyme IIAGlc has been unexpectedly implicated in some peculiar functions (Visick KL, 2007). In A. fischeri ES114, bioluminescence was found to be regulated by CRP-cAMP (Lyell NL, 2013). In Vibrio vulnificus, the TonB3 system is CRP-dependent (Alice AF, 2012).
Pasteurella multocida adenylate cyclase was shown to be regulated by Escherichia coli Enzyme IIAGlc (Mock M, 1991). Thus, the E. coli regulation of the cAMP levels most likely occurs in P. multocida. Incidentally, the cya gene of P. multocida was detected in all tested subspecies and serogroups of P. multocida (Escande F, 1993).
In Haemophilus influenzae, cAMP is essential to competence for DNA transformation (Dorocicz IR, 1993). 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 (Gwinn ML, 1996). The report that addition of cAMP reverses the effect of PTS mutations indicates an E.coli-like regulation of adenylate cyclase by the PTS. 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 (Redfield RJ, 2005), and further characterized (Cameron AD, 2008). This regulon was studied in E. coli and other Gammaproteobacteria (Cameron AD, 2006). Interestingly, competent genes in E. coli were also regulated by CRP-cAMP (Sinha S, 2009) however physiological conditions leading to competence in E. coli have not yet been defined (Jaskólska M, 2015). Unlike E. coli under laboratory conditions, cyclic AMP phosphodiesterase was also involved in regulating the cAMP level (Macfadyen LP, 1998).
Experimental data are not presently available for Pseudomonas aeruginosa as regards the regulation of adenylate cyclase. Visit the Pseudomonas Genome Database and search 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 (Smith RS, 2004). In fact, P. aeruginosa membrane-bound adenylate cyclase (encoded by cyaB) is more likely to play a prominent role in virulence (Lory S, 2004). Unlike E. coli, cAMP levels in P. aeruginosa do not vary significantly with the carbon source used in the culture medium (Siegel LS, 1977). Also, even though the cAMP receptor of P. aeruginosa (Vfr) is a global regulator of transcription (Kanack KJ, 2006), it does not play a role in catabolite repression (Suh SJ, 2002), as previously inferred (Phillips AT, 1981). A small RNA (CrcZ) has been actually described as global regulator of catabolite repression (Sonnleitner E, 2009). These results however do not rule out a regulatory mechanism for P. aeruginosa adenylate cyclase possibly involving the 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.
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, and 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. All IIC domains of the V. cholerae PTS were deleted to investigate the role of PTS transport in infection (Hayes CA, 2017b, Comment). However, regulations by PTS transport, i.e., regulation of cAMP levels and inducer exclusion, were most likely eliminated, as Enzyme IIAGlc remained phosphorylated thus possibly maintaining a relatively high and constant level of cAMP.
