Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegra dation of aromatic compounds

Supporting informationThe metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP+ -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds.This work was supported by the EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797) and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), Contracts of the European Union, and the CAMBIOS (RTC-2014-1777-3) and CONTIBUGS (PCIN-2013-040) projects of the Spanish Ministry of Economy and Competitiveness.Peer reviewe