Abstract
The class Ia ribonucleotide reductase (RNR), encoded by the nrdAB operon in Pseudomonas aeruginosa, has a long 5' untranslated region (5' UTR) whose regulatory role remains poorly understood. In this study, we investigated the functional significance of the nrdAB 5' UTR using a comprehensive set of bioinformatic and experimental approaches, including gene expression profiling, relative protein levels, and in vivo infection assays in the Galleria mellonella animal model. Our results demonstrate that the 5' UTR negatively regulates nrdA expression by reducing transcript levels, decreasing mRNA stability and limiting protein abundance. Truncation of this region led to increased expression, particularly during the stationary phase, suggesting that this region may contribute to modulating RNR activity during the stationary phase, when dNTP demand is reduced. In vivo, the Δ5' UTR mutant exhibited reduced virulence compared to the wild-type strain, accompanied by elevated nrdA mRNA levels and a modest decrease in nrdJ expression. Although these changes may contribute to altered RNR regulation during infection, they are unlikely to fully explain the observed phenotype, indicating that additional factors may be involved. Although no specific regulatory elements within or acting upon the 5' UTR were probed under the tested conditions, the possibility of such mechanisms cannot be excluded, and further studies will be needed to elucidate the underlying molecular mechanisms. Overall, our findings demonstrate that the 5' UTR plays a regulatory role in modulating nrdAB operon expression and contributes to maintaining RNR system homeostasis in P. aeruginosa.