Low CyaA expression and anti‐cooperative binding of cAMP to CRP frames the scope of the cognate regulon of Pseudomonas putida

Although the soil bacterium Pseudomonas putida KT2440 bears a bona fide adenylate cyclase gene (cyaA), intracellular concentrations of 30,50-cyclic adenosine monophosphate (cAMP) are barely detectable. By using reporter technology and direct quantification of cAMP under various conditions, we show that such low levels of the molecule stem from the stringent regulation of its synthesis, efflux and degradation. Poor production of cAMP was the result of inefficient translation of cyaA mRNA. Moreover, deletion of the cAMP-phosphodiesterase pde gene led to intracellular accumulation of the cyclic nucleotide, exposing an additional cause of cAMP drain in vivo. But even such low levels of the signal sustained activation of promoters dependent on the cAMP-receptor protein (CRP). Genetic and biochemical evidence indicated that the phenomenon ultimately rose from the unusual binding parameters of cAMP to CRP. This included an ultratight cAMP-CrpP. putida affinity (KD of 45.0   3.4 nM) and an atypical 1:1 effector/ dimer stoichiometry that obeyed an infrequent anticooperative binding mechanism. It thus seems that keeping the same regulatory parts and their relational logic but changing the interaction parameters enables genetic devices to take over entirely different domains of the functional landscape.The Novo Nordisk Foundation (Grants NNF10CC1016517 and NNF18OC0034818Danish Council for Independent Research (SWEET, DFFResearch Project 8021-00039B)Fondos de la Unión Europea/[RTI2018-095584-B-C42]/FEDER/EspañaEuropean Research Area CoBioTech/[ERA-COBIOTECH 2018 - PCI2019-111859-2]/ERA CoBioTech/Unión EuropeaMinisterio de Ciencia e Innovación/[(H2020-FET-OPENRIA-2017-1-766975]/MCIN/EspañaMinisterio de Ciencia e Innovación/[H2020-NMBP-BIO-CSA- 2018-820699]/MCIN/EspañaMinisterio de Ciencia e Innovación/[H2020-NMBP-TR-IND/ H2020-NMBP-BIO-2018-814650]/MCIN/EspañaMinisterio de Ciencia e Innovación/[MIX-UP H2020-BIO-CN-2019-870294]/MCIN/EspañaIngeniería Microbiana, Salud y Calidad de Vida/[S2017/BMD-3691]/InGEMICS-CM/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA