FleQ-Dependent Regulation of the Ribonucleotide Reductase RepressornrdRinPseudomonas aeruginosaDuring Biofilm Growth and Infection

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 asPseudomonas 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 inP. aeruginosaby 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 hownrdRitself is transcriptionally regulated. In this study, we employed a bioinformatics approach to identify potential transcription factors (TF) involved innrdRregulation. We combined this with promoter-probe vectorsnrdRpromoter fusions to investigatenrdRtranscriptional regulation and identify TFs that modulate its expression in vitro. Our analysis identified four potential TF that could regulatenrdR,and we experimentally confirmed that FleQ is responsible for regulatingnrdRexpression under aerobic and anaerobic conditions. Furthermore, we explorednrdRregulation under biofilm-forming conditions and in theGalleria mellonellainfection model to gain insights into hownrdRmight be regulatedin vivo.