The genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaA(P. putida) gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaA(P. putida) was poorly translated or rendered non-functional in the heterologous host. Yet, generation of cAMP by CyaA(P. putida) could be verified by expressing the cyaA(P. putida) gene in a hypersensitive E. coli strain. On the other hand, the crp(P. putida) gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress-endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp-cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co-opted for a new use.
