Regulatory exaptation of the catabolite repression protein (Crp)–cAMP system in Pseudomonas putida

Abstract

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.

Involvement of the Global Crp Regulator in Cyclic AMP-Dependent Utilization of Aromatic Amino Acids by Pseudomonas putida

“…We therefore concluded that the crp gene product is involved in the utilization of these aromatic amino acids as nitrogen sources. These results are in agreement with those reported by Milanesio et al (27), which showed that crp mutations do not affect the panel of C compounds that can be used by P. putida as growth substrates and that a crp mutant was impaired in the use of certain dipeptides as an N source.…”

“…In contrast with other microorganisms, it should be noted that the characterization of a P. putida crp-deficient mutant revealed that the profile of C sources used by this mutant is identical to that of the wild-type strain (8,27). In P. putida, the crp gene product (PP0424) is annotated as a cyclic AMP (cAMP) receptor protein, with high similarity to Crp of Escherichia coli (62% identity) and Vfr of Pseudomonas aeruginosa (82% identity).…”

“…Utilization of certain D-and L-amino acids by P. putida appears to involve the global regulator Crp, since growth with D-and L-lysine, D-alanine, and L-phenylalanine as N sources was compromised in crp mutants (8,27). In contrast with other microorganisms, it should be noted that the characterization of a P. putida crp-deficient mutant revealed that the profile of C sources used by this mutant is identical to that of the wild-type strain (8,27).…”

Biología sintética: la ingeniería al asalto de la complejidad biológica

“…Por ejemplo, el plumaje apareció en su momento como un mero aislamiento térmico pero luego se transformó en un componente esencial del vuelo de los pájaros. El análisis de los genomas bacterianos proporciona incontables ejemplos de proteínas que nacen para hacer algo que luego resulta ser muy distinto de aquello para lo que finalmente se utilizan (Milanesio et al, 2011). Este proceso, que en Biología evolutiva se denomina exaptación, tiene también incontables equivalentes en ingeniería: un dispositivo que se inventa con un propósito muy específico reaparece con pequeñas modificaciones en otro sitio con una finalidad inesperada e incluso opuesta a la inicial.…”

Regulation of Superoxide Dismutase Synthesis in Humcola lutea Cells under Cu2+ Stress Conditions

“…As it is known, in many cases catabolite repression in microorganisms is correlated with lowered levels of cAMP [ 8,12,13 ]. Our present results (Table 1) illustrate the reversal effect of cAMP on SOD synthesis by H. lutea cells in the presence of glucose.…”