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

Alejandro Arce-Rodríguez, Pablo I. Nikel, Belén Calles, Max Chavarría, Raúl Platero, Tino Krell, Victor de Lorenzo*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

Abstract

Although the soil bacterium Pseudomonas putida KT2440 bears a bona fide adenylate cyclase gene (cyaA), intracellular concentrations of 3′,5′-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 anti-cooperative 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.

Original languageEnglish
JournalEnvironmental Microbiology
Volume23
Issue number3
Pages (from-to)1732-1749
ISSN1462-2912
DOIs
Publication statusPublished - Mar 2021

Bibliographical note

Funding Information:
Authors are indebted to Mario Menc?a for plasmids and other materials, to Juhyun Kim for help in the analysis of RNA-seq transcriptomic data, to Rosa Sedano for chemical analyses and Hortensia Silva and Cristina Garc?a Fontana for assistance with the microcalorimetry experiments. This work was funded by the SETH (RTI2018-095584-B-C42; MINECO/FEDER) and SyCoLiM (ERA-COBIOTECH 2018 - PCI2019-111859-2) Projects of the Spanish Ministry of Science and Innovation, the MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018-820699), SynBio4Flav (H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650) and MIX-UP (MIX-UP H2020-BIO-CN-2019-870294) Contracts of the European Union and the InGEMICS-CM (S2017/BMD-3691) Project of the Comunidad de Madrid (European Structural and Investment Funds-FSE, FECER). P.I.N. gratefully acknowledges the financial support of the The Novo Nordisk Foundation (Grants NNF10CC1016517 and NNF18OC0034818) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. was supported by the Plan Nacional de I + D + I (Grant BIO2010-16937) and the Plan Estatal de Investigaci?n Cient?fica y T?cnica y de Innovaci?n (BIO2016-76779-P). R.P. acknowledges financial support by Uruguayan Agency for Investigation and Innovation (ANII), Research projects: FCE_1_2017_1_136082 and FCE_1_2019_1_156520.

Funding Information:
Authors are indebted to Mario Mencía for plasmids and other materials, to Juhyun Kim for help in the analysis of RNA‐seq transcriptomic data, to Rosa Sedano for chemical analyses and Hortensia Silva and Cristina García Fontana for assistance with the microcalorimetry experiments. This work was funded by the SETH (RTI2018‐095584‐B‐C42; MINECO/FEDER) and SyCoLiM (ERA‐COBIOTECH 2018 ‐ PCI2019‐111859‐2) Projects of the Spanish Ministry of Science and Innovation, the MADONNA (H2020‐FET‐OPEN‐RIA‐2017‐1‐766975), BioRoboost (H2020‐NMBP‐BIO‐CSA‐2018‐820699), SynBio4Flav (H2020‐NMBP‐TR‐IND/H2020‐NMBP‐BIO‐2018‐814650) and MIX‐UP (MIX‐UP H2020‐BIO‐CN‐2019‐870294) Contracts of the European Union and the InGEMICS‐CM (S2017/BMD‐3691) Project of the Comunidad de Madrid (European Structural and Investment Funds‐FSE, FECER). P.I.N. gratefully acknowledges the financial support of the The Novo Nordisk Foundation (Grants NNF10CC1016517 and NNF18OC0034818) and the Danish Council for Independent Research (SWEET, DFF‐Research Project 8021‐00039B). T.K. was supported by the Plan Nacional de I + D + I (Grant BIO2010‐16937) and the Plan Estatal de Investigación Científica y Técnica y de Innovación (BIO2016‐76779‐P). R.P. acknowledges financial support by Uruguayan Agency for Investigation and Innovation (ANII), Research projects: FCE_1_2017_1_136082 and FCE_1_2019_1_156520.

Publisher Copyright:
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.

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