The long 5′UTR of nrdAB modulates transcription, mRNA stability, and virulence in Pseudomonas aeruginosa

The class Ia ribonucleotide reductase ( nrdAB ) operon in Pseudomonas aeruginosa encodes a long 5’ untranslated region (5’UTR), whose regulatory role remains poorly understood. In this study, we studied the functional significance of the nrdAB 5’UTR through a combination of comprehensive set of bioinformatic and experimental approaches combining gene expression studies, protein analysis, and infection in Galleria mellonella in vivo animal model. Our results demonstrate that the 5’UTR negatively regulates nrdA expression by reducing transcription and decreasing mRNA stability. Deletion of the 5’UTR led to increased nrdA mRNA and protein levels, particularly during stationary phase, suggesting a role in downregulating ribonucleotide reductase activity when dNTP synthesis is no longer required. Structural predictions indicated that the absence of the 5’UTR may lead to a more flexible mRNA conformation, which could facilitate ribosome accessibility. Notably, this deregulation disrupted the balance between nrdA and nrdJ expression, leading to reduced virulence in a G. mellonella infection model. This effect results from a slight decrease in nrdJ expression combined with an increase in nrdA mRNA levels, which may compromise the optimal RNR regulation during infection. These findings highlight the 5’UTR as a key regulatory element in fine-tuning nrdAB expression and maintaining RNR system homeostasis in P. aeruginosa.