FleQ-Dependent Regulation of the Ribonucleotide Reductase Repressor nrdR in Pseudomonas aeruginosa During Biofilm Growth and Infection

Abstract

Ribonucleotide reductases (RNRs) are essential enzymes involved in DNA synthesis and repair, catalyzing the conversion of ribonucleotides to deoxyribonucleotides (dNTPs). While all living cells possess at least one RNR encoded in their genome, certain organisms, such as Pseudomonas aeruginosa, encode multiple RNR classes. This multiplicity provides a competitive advantage, allowing these organisms to adapt and colonize different environments. Despite their importance, the mechanisms coordinating the expression of different RNRs in microorganisms with multiple RNR classes remain poorly understood. The transcriptional regulator NrdR has been implicated in controlling the expression of all three RNR classes in P. aeruginosa by binding to conserved NrdR boxes within the promoter regions of the RNR genes. To gain insights into the regulation of the different RNR genes, it is first necessary to understand how nrdR itself is transcriptionally regulated. In this study, we employed a bioinformatics approach to identify potential transcription factors (TF) involved in nrdR regulation. We combined this with promoter-probe vectors nrdR promoter fusions to investigate nrdR transcriptional regulation and identify TFs that modulate its expression in vitro. Our analysis identified four potential TF that could regulate nrdR, and we experimentally confirmed that FleQ is responsible for regulating nrdR expression under aerobic and anaerobic conditions. Furthermore, we explored nrdR regulation under biofilm-forming conditions and in the Galleria mellonella infection model to gain insights into how nrdR might be regulated in vivo. ImportanceThis study reveals a nuanced regulatory mechanism by which the transcription factor FleQ, modulated by intracellular c-di-GMP levels, governs the expression of the essential gene nrdR in Pseudomonas aeruginosa. By demonstrating that FleQ acts as an activator under planktonic and infection conditions and as a repressor during biofilm formation, the findings reveal a dual regulatory role that aligns with the bacteriums transition between acute and chronic infection states. This dynamic control of nrdR, a key repressor of ribonucleotide reductases, links environmental sensing to nucleotide metabolism, offering new insights into how P. aeruginosa adapts to diverse and hostile environments. These results not only deepen our understanding of bacterial gene regulation but also highlight potential targets for disrupting biofilm-associated persistence in clinical settings.