Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

Abstract

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

Differential adaptability between reference strains and clinical isolates of Pseudomonas aeruginosa into the lung epithelium intracellular lifestyle

“…Although hypoxia was confirmed to occur in the different infections, possible trace oxygen present in different intracellular compartments may prevent the activity of the anaerobic class III RNR. The oxidative stress present in the cellular compartments promoted by infections, as has been observed in CF-affected lungs, may induce the expression of class II RNR [34]. Hence, we hypothesize that the oxygen independence of class II RNR in addition to the stress present in the infectious environment favor the expression of this RNR class and the subsequent adaptation of P. aeruginosa to the intracellular lifestyle, which allows the formation of a bacterial reservoir and contributes to infection recurrence and chronicity.…”

Metabolic manipulation by Pseudomonas fluorescens: a powerful stratagem against oxidative and metal stress

“…To combat this situation, P. fluorescens elaborates a detoxification strategy reliant on the conversion of the divalent metal into calcite, a crystalline insoluble calcium carbonate. To achieve this metabolic feat, the microbe initially immobilizes the Ca in an epicellular organic matrix where the presence of the up-regulated carbonic anhydrase enables the controlled formation of calcite where the toxic divalent metal is locked [62][63][64][65].…”

Monitoring Gene Expression during a Galleria mellonella Bacterial Infection

“…G. mellonella larvae were injected with an infective dose of P. aeruginosa PAO1 wild-type and P. aeruginosa PAO1 containing plasmids derived from pETS130 ( Table S1 ), which encode transcriptional fusions of the nrdA and nrdJ promoters with GFP and E2Crimson that were previously constructed in our laboratory (pETS134, pETS180, pETS226, and pETS227) [ 14 , 19 ]. As a control, a group of larvae was only injected with 1× PBS (Fisher Scientific, Madrid, Spain).…”