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

The long 5′UTR of nrdAB modulates transcription, mRNA stability, and virulence in Pseudomonas aeruginosa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The long 5′UTR of nrdAB modulates transcription, mRNA stability, and virulence in Pseudomonas aeruginosa Ángela Martínez-Mateos, Alba Rubio-Canalejas, Lucas Pedraz, Eduard Torrents This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6937143/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract 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. Biological sciences/Microbiology/Bacteria/Bacterial genetics Biological sciences/Microbiology/Bacteria/Bacterial physiology Biological sciences/Microbiology/Bacteria/Bacterial transcription ribonucleotide reductase 5’UTR mRNA stability transcription protein levels Galleria mellonella Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen that exhibits a notable metabolic flexibility, genomic variability and phenotypic diversity 1 . Due to its ability to survive in diverse environments —along with its both acquired and innate antibiotic resistance mechanisms— and its capability to form biofilms, there is an urgent need for the discovery of new therapeutic strategies 1 , 2 One promising therapeutic target is ribonucleotide reductase (RNR), a family of metalloenzymes responsible for the de novo synthesis of deoxyribonucleotides (dNTPs) through a radical-based chemistry mechanism 3 . Three main classes of RNRs have been described: class I (subtypes Ia, Ib, Ic, Id and Ie) 4 , Class II and Class III 3 . This classification is based on structural differences, oxygen needs, metallocofactor requirements and the mechanism used for their radical generation 3 , 5 , 6 . P. aeruginosa is one of the few organisms known to encode all three main classes, reflecting the finely tuned regulatory systems of this microorganism 6 . Although class I RNRs are among the best-characterized enzymes, little is known about class Ia in P. aeruginosa 7 . This RNR class consists of two homodimeric subunits (α and β), encoded by the nrdAB operon. So far, two transcriptional factors (AlgR and NrdR) have been identified as regulators of the operon 6 , 8 , 9 . However, key aspects of the regulatory networks of nrdAB operon remain unclear. Several years ago, a 5’ untranslated region (5’UTR) upstream nrdA was predicted through bioinformatic analysis 8 , but further experimental validation is needed to confirm its structure and regulatory role. The 5’UTRs of bacterial mRNAs have increasingly been recognized as crucial regulatory elements that influence gene expression at multiple levels. Rather than being passive sequences between promoters and start codons, 5’UTRs harbor a wide variety of regulatory elements that actively control translation efficiency, transcript stability and transcription termination 10 , 11 . Their functional diversity includes ribosome binding sites (RBS) or Shine-Dalgarno (SD) sequences, upstream open reading frames (uORFs), binding motifs for small RNAs (sRNAs), and dynamic secondary structures capable of responding to environmental or metabolic signals, among others 12 , 13 . Changes in 5’UTR sequence or structure can alter gene expression by affecting ribosome accessibility, modulating mRNA degradation, or engaging in complex networks with trans-acting elements like sRNAs and RNA-binding proteins 14 , 15 . For example, riboswitches within 5’UTRs bind metabolites, ions, or coenzymes to control downstream gene expression 11 , 16 . Given that transcription and translation are tightly coupled in bacteria and they occur in the cytoplasm, the regulatory information embedded in 5’UTRs plays a particularly prominent role in post-transcriptional control 11 . Notably, 5’UTRs do not function independently, as recent studies suggest that riboswitches, sRNAs, antisense RNAs, and 5’UTRs themselves can regulate one another, forming intricate regulatory networks 15 . Despite their importance, many 5’UTRs remain poorly characterized, and their potential as tunable regulatory modules in bacterial gene expression is only beginning to be uncovered 13 . For this reason, the aim of this study is to understand the nature and functional role of the predicted 5’UTR of nrdAB operon in P. aeruginosa. By exploring its potential impact on the regulation of nrdAB , we seek to determine whether it influences transcript stability, translation efficiency or gene expression dynamics under different conditions. Results and Discussion The nrdAB operon encodes a long 5’ untranslated region (5’UTR) with an unknown function. The oxygen-dependent class Ia ribonucleotide reductase (RNR) of Pseudomonas aeruginosa is encoded by two cotranscribed genes, nrdA and nrdB 34 . In a previous study carried out by our group, several regulatory features of the nrdAB operon were identified. On one hand, transcriptional binding sites for AlgR and NrdR were identified 6 , 8 (Fig. 1 a). On the other hand, bioinformatic analyses revealed that the transcription starting site (TSS) of nrdAB was located approximately 400 bp upstream of the translational start codon (ATG), indicating the presence of a long 5’UTR 6 (Fig. 1 a-b, yellow triangle). Based on this information, the putative − 10 and − 35 promoter boxes of the nrdAB operon were mapped (Fig. 1 a). To experimentally validate the transcription start site of the nrdAB mRNA, we combined insights from laboratory experiments and genome annotation databases, which consistently indicated that the TSS is located between 408 and 411 bp upstream of the translational start site (Fig. 1 a-b). A primer extension assay confirmed that the 5’ end of the nrdAB transcript begins with a cytosine nucleotide located 411 bp upstream of the predicted translational start site (Fig. 1 a-c, blue triangle), while 5’RACE positioned the TSS 409 bp upstream (Fig. 1 a-b, d, pink triangle) corroborating the presence of a long 5’UTR. Given that bacterial 5’ UTR are typically short (20–30 nucleotides), the presence of such an extended 5’UTRs suggests a potential regulatory element involved in modulating nrdA expression 35 . The 5’UTR does not appear to contain a cobalamin riboswitch. Long 5’UTRs have been associated with several regulatory elements 35 , 36 . Among these are riboswitches, the vast majority of which are located in the 5’UTRs of mRNA 37 . A bioinformatic analysis using Riboswitch Scanner identified a potential cobalamin riboswitch within its 5’UTR (Supplementary Figure S1 a). Notably, the nrdABS operon, which encodes the class Ia RNR in Streptomyces , is regulated by a cobalamin riboswitch 38 . This is not an isolated case, several RNRs, including the class Ia enzyme from Bacillus halodurans , are also regulated by B 12 riboswitches 39 . Given this context, it was reasonable to investigate the presence of a similar regulatory element in the 5’UTR of the P. aeruginosa nrdA gene. Furthermore, a known connection exists between vitamin B 12 and RNR activity, as demonstrated by the class II RNR from Pseudomonas aeruginosa , which, like other class II RNRs, relies on the availability of S-adenosylcobalamin (vitamin B 12 ) 18 . Cobalamin riboswitches constitute a diverse family characterized by a highly conserved secondary structure and a sequence signature known as the B 12 box 40 . To investigate the presence of a putative B 12 box, a multiple sequence alignment was performed between the predicted P. aeruginosa nrdA cobalamin riboswitch and various P. aeruginosa cobalamin riboswitches obtained from the RegPrecise database 24 (See Materials and Methods). As shown in Fig. 2 a, the alignment revealed a conserved putative B 12 box and structural similarities between the predicted sequence alignment from 41 , and the nrdA sequence (PA1156) from P. aeruginosa . To validate the presence of a functional B 12 riboswitch, transcriptional and translational GFP fusions of nrdA were constructed (see Materials and Methods), and GFP reporter assays were performed (see Materials and Methods). These fusions served as B 12 -sensors to evaluate the presence of a functional riboswitch and the effect of coenzyme B 12 on downstream gene expression. As shown in Fig. 2 b-c, the assays did not demonstrate a significant difference in GFP expression across increasing concentrations of coenzyme B 12 in either the transcriptional (Fig. 2 b) or translational (Fig. 2 c) fusions. While the precise mechanisms of coenzyme B 12 binding and downstream regulation remain poorly understood 42 , translational regulation appears to be the most prevalent mechanism among cobalamin riboswitch in γ-Proteobacteria 41 . Moreover, it has been shown that that cobalamin riboswitches are a complex family capable of sensing and responding to a range of corrinoid compounds 43 . However, some cobalamin riboswitches are known to respond only to specific subsets of these compounds. In addition, the sequence and structure of the nrdA promoter sequence did not perfectly align with those of experimentally validated B 12 riboswitches (Fig. 2 a). In conclusion, although bioinformatic analysis initially predicted the presence of a cobalamin riboswitch in the nrdAB 5' UTR, our experimental results do not support a functional role for coenzyme B 12 in regulating nrdAB expression. Therefore, we cannot conclude that the 5’UTR of nrdAB functions as a cobalamin riboswitch. The 5’UTR does not encode an upstream open reading frame (uORF) or a small RNA (sRNA). Other regulatory elements commonly found within 5’UTR include upstream ORFs (uORFs), which are short open coding sequences often involved in post-transcriptional regulation through ribosome-mediated translational attenuation 11 . To assess the non-coding nature of the nrdAB 5' UTR, both bioinformatic and experimental approaches were employed. As shown in Fig. 3 a, the full 411 bp 5’UTR sequence was analysed using the Coding Potential Calculator 2 (CPC2) tool 27 , which predicted a 49 amino acid peptide. However, the coding probability was low, and a BLASTp search against the non-redundant protein database (nr) revealed no significant similarity 26 . Similarly, a translated BLAST (tBlastn) search against the core nucleotide database (core nt) yielded less than 30% coverage to hypothetical proteins from various P. aeruginosa strains 26 . To further confirm the untranslated nature of the 5’UTR, the entire sequence was cloned downstream of an arabinose-inducible promoter plasmid (See Materials and Methods). Theoretically, the 5’ UTR could encode a protein of approximately 15 kDa; in parallel, the predicted 49-amino acid peptide would correspond to a protein of less than 10 kDa. However, as shown in Fig. 3 b, no protein bands of either size (10 or 15 kDa) were detected upon arabinose induction, and protein expression profiles under induced and non-induced conditions were identical. These results suggest that the 5’ UTR does not encode a functional uORFs. Another class of regulatory elements frequently found in 5’ UTR are small RNAs (sRNAs), which are non-coding RNAs that can influence transcription, translation, mRNA stability, and DNA maintenance or silencing 11 , 44 . Most sRNAs are located in intergenic regions (IgRs), transcribed from their own promoter or nearby gene promoters, and often terminate via Rho-independent mechanisms 45 , 46 . To explore this possibility, we analysed the regions upstream and downstream of nrdA for additional TSSs, annotated sRNAs, and Rho-independent terminators. The complete 5’ UTR sequence was submitted to the Rfam 28 and RNAcentral 29 database. No annotated non-coding RNA matching this sequence was identified. Promoter prediction tools such as PromoterHunter 31 and BPROM 32 revealed two possible promoter regions: one previously identified (Fig. 1 a, purple underlined sequences; Fig. 3 c, P1 black arrow), and a second located 226 bp upstream of the nrdA start codon (Fig. 3 c, P2 grey arrow). Analysis using ARNold software 30 identified only the known Rho-independent terminator at the end of the nrdAB operon (Fig. 3 c). Additionally, it has been reported a putative sRNA, pant125, on the negative strand between coordinates 1254329–1254720, overlapping the nrdAB promoter region 47 (Fig. 3 c). However, their RNA-seq analysis, which assumed all non-coding regions correspond to intergenic regions, may have underestimated the functional relevance of 5’UTRs. Since no additional TSSs or terminators were found near the pant125 region (Fig. 3 c), one possible explanation is that pant125 is a product of nrdA transcript processing by RNases, resulting in two transcripts: nrdA mRNA and the putative sRNA. In P. aeruginosa , RNA degradation and processing are mediated by several enzymes, including RNase E ( rne , PA2976), ATP-dependent RNA helicase RhlB ( rhl , PA3861) and RNAse R ( rnr , PA4937), among others (KEGG pathway: pae03018) 48 . To determine whether one of these RNA processing enzymes might be responsible for processing the nrdA transcript, we conducted GFP transcriptional reporter assays comparing nrdA expression (with and without 5’UTR region) in P. aeruginosa PAO1 wild-type and RNA processing enzymes mutant strains (Supplementary Figure S2). However, none of the RNA processing enzymes mutants showed increased nrdA expression compared to the wild-type strain (Supplementary Figure S2a). Furthermore, when the nrdA promoter (P nrdA ) lacking the 5’ UTR was tested, differences in expression between strains persisted (Supplementary Figure S3b). Theoretically, if a specific RNA processing enzymes cleaved nrdA mRNA via 5’UTR-dependent mechanisms, such differences should disappear in the absence of the 5’ UTR. The fact that they did not, combined with the pleiotropic effects of the RNase mutations, complicates the identification of a specific regulatory mechanism. In conclusion, the combined bioinformatic and experimental evidence suggests that the nrdA 5’ UTR does not encode a functional uORF or a regulatory sRNA. The 5’UTR is involved in nrdA regulation by enhancing its transcription. As mentioned earlier, although known transcriptional regulators of nrdAB , such as AlgR and NrdR, have been described 6 , 8 , important gaps remain in our understanding of both the transcriptional and post-transcriptional regulation of nrdAB , particularly concerning the role of the 5’UTR. The significance of the 5’ UTR in nrdAB expression was initially assessed through nrdA transcriptional fusions of PnrdA with GFP, either including or excluding the 5’UTR (See Materials and Methods). As shown in Fig. 4 a, under aerobic conditions, the presence of the 5’UTR (P nrdA ) resulted in reduced nrdA expression compared to the construct lacking the 5’UTR (P nrdA -∆5’UTR), which showed significantly higher expression. Interestingly, under anaerobic conditions (Fig. 4 a), even though class Ia is enzymatically active primarily under aerobic conditions 3 , 5 the 5’ UTR exerted a similar repressive effect on nrdA expression. Furthermore, transcriptional fusion experiments revealed growth-phase-dependent differences in expression, also influenced by the presence of the 5’ UTR. For constructs containing the 5’UTR (P nrdA ), expression decreased during the stationary phase compared to the exponential phase under both aerobic and anaerobic conditions. In contrast, the P nrdA -∆5’UTR fusion showed increased expression during the aerobic stationary phase relative to exponential growth. However, this increase was not observed under anaerobic conditions, where expression decreased during the stationary phase, mirroring the pattern seen with the P nrdA construct. To validate these findings in a more physiological context, nrdA expression was compared between P. aeruginosa PAO1 wild-type and a specific chromosomal deletion mutant generated using the CRISPR-cas9 system 20 , lacking the nrdA 5’UTR (PAO1 ∆5’UTR) (See Materials and Methods). As shown in Fig. 4 b, nrdA expression was consistently higher in the ∆5’UTR mutant compared to the wild-type strain, under both aerobic and, especially, anaerobic conditions, corroborating the results obtained with the GFP reporter assay. Additionally, expression dynamics across growth phases reflected those observed with the plasmid-based reporter system: in PAO1 WT, nrdA expression was higher during the exponential phase than in the stationary phase under both aerobic and anaerobic conditions. However, in PAO1 ∆5’UTR, nrdA expression remained relatively stable across exponential and stationary growth phases, regardless of oxygen availability (Fig. 4 c). As simplified model is presented in Fig. 4 d. Under aerobic conditions, both approaches, GFP transcriptional fusion and qRT-PCR, were consistent: nrdA expression was higher in the absence of the 5’UTR and increased progressively throughout bacterial growth phases. Under anaerobic conditions, although nrdA expression without 5’ UTR was also higher during the stationary phase, a discrepancy emerged between the two methods. In the GFP transcriptional assay, expression appeared to decrease in the stationary phase, whereas qRT-PCR quantifies mRNA levels directly, showed increased expression. This difference may be attributed to the nature of the assays, the plasmid-based reporter system measures expression indirectly, while the chromosomal mutant reflects a more physiologically relevant context. Thus, the latter is considered more reliable. In summary, these findings highlight the regulatory complexity of class Ia RNR in P. aeruginosa , which is influenced by both growth phase and oxygen availability. The data suggest that the 5’UTR of nrdA plays a key role in downregulating transcription and reducing RNR protein expression and enzymatic activity during growth, particularly in stationary phases when dNTPs synthesis is no longer required due to the cessation of DNA replication. The 5’UTR influences nrdAB stability and decay. As shown in Fig. 4 , the absence of the 5’UTR appears to increase nrdA transcription and, consequently, translation. 5’UTRs are known to harbour multiple regulatory elements that can contribute to post-transcriptional regulation of the nrdAB operon 11 , 14 . One such regulatory mechanism involves mRNA stability and decay. To investigate the mRNA half-life of nrdAB , we employed two experimental approaches using the same methodology (See Materials and Methods). In the first approach, we indirectly estimated nrdA half-life using a plasmid-based system, where the cat gene (chloramphenicol acetyltransferase) served as a reporter for nrdA expression. mRNA decay was analysed using first-order kinetics (See Materials and Methods). In the second approach, we measured nrdA half-life from the chromosomal copy of nrdAB in both P. aeruginosa PAO1 wild-type and PAO1 Δ5’UTR strains (See Materials and Methods). As shown in Fig. 5 a, the degradation rate (K) of nrdA transcript lacking the 5’UTR (WT + P nrdA -Δ5’UTR) was slower, with a best fit value of 0.07 min − 1 (95% CI: 0.03768 to 0.1352 min − 1 ) compared to the wild-type construct (WT + P nrdA ) which has a 0.21 min − 1 decay rate (95% CI: 0.1876 to 0.2329). Specifically, best-fit half-life values were 3.32 min (95% CI: 2,976 to 3,695 min) for wild-type nrdA and 9.53 minutes (95% CI: 5.129 to 18.39 min) for nrdA without the 5’UTR, indicating that the latter is approximately 3 times more stable (Fig. 5 a and c). Similarly, chromosomal nrdA measurements (Fig. 5 b) revealed a comparable trend. In the P. aeruginosa PAO1 Δ5’UTR, nrdA mRNA exhibited a reduced decay rate of 0.83 min − 1 (95% CI: 0.7624 to 0.8988 min − 1 ) compared to the wild-type which has a decay rate of 1.5 min − 1 (95% CI: 1.404 to 1.603 min − 1 ). The best-fit half-life values were 0.46 min for the wild-type (95% CI: 0.4323 to 0.4938 min) and 0.84 min for the Δ5’UTR (95% CI: 0.7712 to 0,9091 min), suggesting approximately a 2-fold increase in stability (Fig. 5 b and c). To confirm the specificity of this effect, we analysed the expression of nrdJ (the class II ribonucleotide reductase from PAO1) as a reference using both approaches. As shown in Fig. 5 a and 5 B, the decay curves for nrdJ were nearly identical across all strains and constructs, and the best-fit half-life values were similarly consistent between strains using both approaches. In the plasmidic one-phase decay (Fig. 5 c) the decay rates (K) were 0.44 min − 1 (95% CI: 0.4361 to 0.4499 min − 1 ) for the WT + P nrdA construct compared to 0.30 min − 1 (95% CI: 0.2618 to 0.3158 min − 1 ) for the WT + P nrdA -Δ5’UTR construct. A similar trend was observed the chromosomal one-phase decay (Fig. 5 d), where the decay rates were 1.96 min − 1 (95% CI: 1.818 to 2.149 min − 1 ) for WT and 2.1 min − 1 for Δ5’UTR (95% CI: 1.263 to 2.913 min − 1 ). Half-life values followed the same pattern: in the plasmidic context (Fig. 5 c), 1.57 min (95% CI: 1.541 to 1.589 min) for WT + P nrdA construct compared to 2.42 min (95% CI: 2.195 to 2.648 min) for WT + P nrdA -Δ5’UTR; and in the chromosomal context (Fig. 5 d), 0.35 min (95% CI: 0.3225 to 0.3813 min) for WT compared to 0.33 min (95% CI: 0.2379 to 0.5490 min) for Δ5’UTR. Differences between the plasmidic and the chromosomal experiments consistently demonstrate that the 5’ UTR specifically influences the half-life and decay of nrdA but does not affect nrdJ . Despite the methodological variations and some statistical limitations in certain cases, the overall trend is maintained across both experimental systems, corroborating the same conclusion regarding the role of the 5’ UTR in nrdA stability. Additionally, bacterial gene expression is influenced not only by the 5’ UTR but also by the coding sequence and downstream reporter gene 10 , 14 . The 5’UTR-lacking nrdA transcript appears more structurally flexible, promoting translation. The core regulatory function of 5’UTRs involves their dynamic secondary structures, which can influence translation attenuation, transcript stability, and ribosome accessibility 11 . Our findings suggest that the nrdAB 5’UTR acts as a regulatory element that balances nrdA expression, as its absence results in increased transcription and translation (Fig. 4 ). This may be attributed to increased mRNA levels, and consequently more protein, as well as slightly increased stability of the ∆5’UTR mRNA (Fig. 5 ). However, we sought to determine whether this effect is also driven by structural differences that promote translation. To address this, we used RNAfold (See Materials and Methods) to predict the secondary structures of nrdAB transcripts with and without the 5’ UTR. As shown in Fig. 6 a, both transcripts exhibit nearly identical minimum free energy values (-1950.10 Kcal/mol for nrdAB and − 1819.6 Kcal/mol for nrdAB without 5’UTR). Figure 6 b presents mountain plots of their optimal predicted structures, with arrows indicating the 5’ and 3’ ends. While the central regions of both transcripts are similar, the 5’ and 3’ ends of the wild-type transcript appear to be more structured and complex compared the ∆5’UTR variant. Western-blot analysis (Fig. 6 c) confirmed that NrdA protein levels are elevated in the Δ5’UTR strain under both aerobic and anaerobic conditions, during both exponential and stationary growth phases, consistent with the transcriptional data (Fig. 4 ). This supports the hypothesis that the more flexible RNA structure of the ∆5’UTR transcript enhances ribosome access and translation efficiency. However, RNAfold predictions showed no significant difference in total free energy of binding at the ribosome binding site (RBS) (Fig. 6 a, -11.46 Kcal/mol for nrdAB and − 11.44 Kcal/mol for nrdAB without 5’UTR), implying similar RBS accessibility. Therefore, the data suggest that the 5’UTR primarily regulates nrdA at the transcriptional level rather than through translational initiation and may be responsible of the down-regulation of nrdA expression during stationary growth, when dNTP synthesis is no longer required. The 5’ UTR modulates P. aeruginosa pathogenicity during Galleria mellonella infection. Previous studies have shown that nrdJ is strongly upregulated during G. mellonella infection, whereas nrdA plays a more minor role 21 .To assess whether the increased nrdA transcription and NrdA protein levels resulting from 5’UTR deletion affects P. aeruginosa PAO1 virulence, we performed infection experiments in G. mellonella . Figure 7 presents virulence analysis using the G. mellonella infection model. In Fig. 7 a Kaplan-Meier survival curves are shown for larvae infected with P. aeruginosa PAO1 wild-type and P. aeruginosa PAO1 Δ5’UTR ( nrdA ) (chromosomal deletion). The curves were significantly different according to the Log-rank (Mantel-Cox) test (* p < 0.05), showing a higher statistically significant Median Survival Time (MST) of G. mellonella larvae infected with P. aeruginosa PAO1 Δ5’UTR (22 hours) compared to those infected with PAO1 WT (20 hours) (Fig. 7 b). In parallel, RT-qPCR was performed to analyse the expression of nrdA in both strains during infection. Samples were collected at 16 h post-infection (See Materials and Methods). Fold Change values were higher in P. aeruginosa PAO1 Δ5’UTR compared to PAO1 WT (Fig. 7 c). We hypothesize that increased nrdA expression in the ∆5’UTR strain does not confer a fitness advantage. Instead, it may disrupt the balance between nrdA and nrdJ expression under both aerobic and anaerobic conditions, at both mRNA and protein levels (see Supplementary Figure S3a and S3c) as well as during infection (see Supplementary Figure S3b). Specifically, nrdJ expression is decreased at the mRNA level under aerobic conditions but not under anaerobic conditions in PAO1 Δ5’UTR (see Supplementary Figure S3a), while NrdJ protein levels appear increased under both aerobic and anaerobic conditions (see Supplementary Figure S3c). Additionally, nrdJ expression seems to be reduced during G. mellonella infection (see Supplementary Figure S3b). Therefore, this imbalance produced by the absence of the 5’ UTR may impair the coordinated regulation of the ribonucleotide reductase (RNR) system between nrdA and nrdJ , rendering the ∆5’UTR strain less fit and, consequently, less virulent (Fig. 8 ). Material and methods Strains, plasmids, and growth conditions The bacterial strains and the plasmids used are listed in Supplementary Table S1 . E. coli and P. aeruginosa strains were routinely grown in Luria-Bertani (Scharlab, Spain) medium or minimal medium (MM) 17 at 37ºC. For anaerobic growth, LB medium containing KNO 3 (10 g/L) (LBN) was used in screw-cap Hungate tubes 18 . Liquid cultures were shaken at 200 rpm. Antibiotics were added at the following concentrations: 50 µg/mL ampicillin and 10 µg/mL gentamicin for E. coli ; and 100 µg/mL gentamicin, 40 µg/mL tetracycline, and 300 µg/mL carbenicillin for P. aeruginosa . For transcriptional and translational assays, rifampicin (Sigma Aldrich, Merck) was used at a final concentration of 200 µg/mL, along with variable concentrations of adenosyl cobalamin or coenzyme B 12 (Sigma Aldrich, Merck), depending on the experiment. DNA manipulation DNA manipulation and plasmid constructions were performed using standard protocols 19 . All kits and molecular biology enzymes used were obtained from Thermo Fisher Scientific (Spain) and were used according to the manufacturer’s instructions. DNA fragments were amplified using Phusion High-Fidelity DNA polymerase or DreamTaq Green PCR MasterMix with the primers listed in Supplementary Table S2. DNA fragments were isolated and purified from agarose gels using a GeneJet Gel Extraction kit (Thermo Fisher Scientific) and Monarch® DNA gel extraction kit (New England Biolabs) in the case of digested DNA. Plasmid DNA was extracted using a GeneJET Plasmid Miniprep kit (Thermo Fisher Scientific) and transferred into P. aeruginosa cells via electroporation with a Gene Pulser XCell electroporator (Bio-Rad) as previously described 18 . All the constructs obtained were verified by DNA sequencing by Eurofins Genomics. Plasmid construction The plasmids pETS257, pETS258, and pETS259 were constructed as follows (See Supplementary Table S1 ). Briefly, the nrdA promoter region was amplified using the primers 1 and 5, for the P nrdA translational fusion (pETS258); 1 and 3, and 2 and 4 for transcriptional P nrdA -Δ5’UTR gfp fusion (pETS257); 1 and 3, and 4 and 5 for P nrdA -Δ5’UTR gfp translational fusion (pETS259). For P nrdA -Δ5’UTR constructs, two fragments were generated from primers described before and used as templates for overlap extension PCR with primers 1 and 2. The GFP gene (green fluorescent protein) was amplified from pETS130 using 7 and 8 primers. The gfp insert, P nrdA and P nrdA -Δ5’UTR inserts were digested with the Sma I restriction enzyme and ligated with T4 ligase to generate P nrdA and P nrdA -Δ5’UTR GFP translational fusions. The resulting fragments (P nrdA -Δ5’UTR, P nrdA -Δ5’UTR:: gfp , P nrdA :: gfp , were cloned separately to pJET 1.2 vector (Thermo Scientific) and transformed into E. coli DH5a. The resulting plasmids and pETS130-GFP vector were digested with Bam HI- Kpn I (translational GFP fusions) or Bam HI- Sma I (transcriptional GFP fusion) and ligated using the T4 ligase enzyme. These plasmids were electroporated into P. aeruginosa PAO1 and transcriptional GFP fusion plasmids were also electroporated into P. aeruginosa Δ rne , P. aeruginosa Δ rhl and P. aeruginosa Δ rnr. The plasmids pJN106 and pJN107 were generated as described below (Supplementary Table S1 ). The 5’UTR forward region was amplified by primers 9 and 10, while the 5’UTR reverse orientation was amplified by primers 11 and 12. The resulting fragments, named 5’UTRfor and 5’UTRrev, were cloned separately to pJET 1.2 and transformed into E. coli DH5a. The resulting plasmids and pJN105 vector were digested with Eco RI- Pst I and ligated using T4 ligase enzyme. These plasmids were electroporated into P. aeruginosa PAO1. CRISPR-cas9 mutant construction P. aeruginosa PAO1 Δ5’UTR ( nrdA ) chromosomal deletion mutant was generated using the CRISPR-cas9 toolkit described before 20 , following the established protocol with some modifications. The recombineering ssDNA oligonucleotide consisted of 50 nucleotides downstream of the transcriptional starting site (located 409 bp upstream of the nrdA start codon-ATG), directly followed by 50 nucleotides downstream of the nrdA ribosome binding site (located 9 bp upstream of the start codon). The oligonucleotide was synthesized by Eurofins Genomics (see Supplementary Table S2). P. aeruginosa PAO1 pSH124 cells were electroporated with both plasmids pS149 (pS148-Δ5’UTR) and the recombineering oligonucleotide (5’UTR_PAO1(+)-Rec) (Supplementary Table S1 and S2). Screening of edited strains was performed using two consecutive colony PCR (cPCR): an initial cPCR was conducted on pools of colonies, and upon identification of a positive pool, individual colonies were subsequently tested by cPCR. Positive colonies were sequenced by Eurofins Genomics, and the plasmids were subsequently cured as described in the protocol. Gene reporter assay experiments Green fluorescent protein reporter assay was carried out with two methodologies; first, P. aeruginosa bacterial pETS130-GFP derivatives growth was monitored by measuring optical density at 600 nm wavelength (OD 600 ) on growth medium (LB for aerobic cultures and LBN for anaerobic cultures) at 37°C and 200 rpm to OD 600 = 0.4–0.7 (Exponential) and OD 600 ~ 2 (Stationary) where three independent 1 ml samples were taken. Samples were centrifuged at 6000 rpm for 10 min. The pellet was washed with phosphate-buffered saline (PBS) (Fisher Scientific) solution containing 2% formaldehyde and stored in the dark at 4°C for 10 min. The samples were centrifuged again, and the pellets were resuspended in 1 mL PBS. The fluorescence of the samples was measured in 96-well plates (Costar ® 96-Well Black polystyrene plate, Corning) in an Infinite 200 Pro Fluorescence Microplate Reader (Tecan, Switzerland). Alternatively, P. aeruginosa bacterial pETS130-GFP derivatives growth and GFP fluorescence was monitored simultaneously every 20–30 minutes in a 96-well plate (Costar ® 96-Well Black polystyrene plate, Corning) in a Spark microplate reader (Tecan, Switzerland) at 37ºC. For the riboswitch test, P. aeruginosa strains containing derivatives of the pETS130-GFP plasmid were grown in minimal medium (MM) 17 until OD 600 ~ 0.3. Different concentrations of B 12 vitamin were added (1 mg/ml, 2.5 mg/mL and 10 mg/mL) and GFP expression was measured in a 96-well plate (Costar ® 96-Well Black polystyrene plate, Corning) every 20 min in an Infinite 200 Pro Fluorescence Microplate Reader (Tecan, Switzerland). Western immunoblot analysis P. aeruginosa PAO1 WT and P. aeruginosa PAO1 Δ5’UTR strain cultures were collected under aerobic and anaerobic conditions during exponential (OD 600 = 0.4–0.7) and stationary (OD 600 ~ 2) growth phases. Protein concentrations were adjusted to 10 µg for all the samples based on quantification using the Bradford Assay (Bio-Rad), with bovine serum albumin (BSA) as the standard. Western blotting was conducted as previously described, with some modifications 5 . A 1:10000 dilution of polyclonal Anti-NrdA or Anti-NrdJ primary antibodies (Agrisera, Sweden; and Thermo Fisher, USA) was used, followed by detection with a donkey anti-rabbit horseradish peroxidase–conjugated secondary antibody (Bio-Rad) at a 1:750 dilution. The antibody–antigen complexes were visualized using Amersham™ ECL™ Prime Western Blotting Reagent (GE Healthcare), according to the manufacturer’s instructions. Protein bands were visualized and analysed by the ImageQuant™ LAS 4000 Mini system (GE Healthcare). The resulting image was processed using Fiji (ImageJ, NIH, USA) for visualization purposes. 5’UTR overexpression P. aeruginosa PAO1 pJN106 and P. aeruginosa PAO1 pJN107 cultures were collected during the exponential phase and after 2 hours of induction with 0.4% L-arabinose. Pellets were lysed with BugBuster® 1X (Sigma Aldrich) according to the manufacturer’s instructions. The lysates were centrifuged at 16000g for 20 minutes at 4 ºC. Protein concentrations were determined by the Bradford assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. A 4–20% Mini-PROTEAN® TGX ™ Precast protein gel (Bio-Rad) was loaded, and the gel was run at 40 mA. RNA isolation, reverse transcription and real-time PCR (qRT-PCR) P. aeruginosa PAO1 strain cultures were collected under aerobic and anaerobic conditions during exponential (OD 600 = 0.4–0.7) and stationary (OD 600 ~ 2) growth phases. Total RNA was extracted using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To further remove DNA contamination, the RNA was treated with TURBO™ DNase (Thermo Fisher Scientific) following the manufacturer’s instructions and verified by PCR with the primers listed in Supplementary Table S2. The amount of RNA was determined using a NanoDrop (Nanodrop spectrophotometer ND-1000). The cDNA retro-transcription was performed using Maxima Reverse Transcriptase (Thermo Fisher Scientific) and Random hexamers (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR measurements were conducted using PowerUp™ SYBR − Green™ (Applied Biosystems) with the SYBR-Green listed in Supplementary Table S2, and detection was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) following the manufacturer’s specifications. The gap gene was used as an internal standard unless otherwise specified. The results were analysed using the ΔΔCt method unless stated differently. Primer extension Bacterial cells were grown in LB medium to an OD 600 of 0.7, and the total RNA was extracted with GeneJET RNA extraction kit (Thermo Fisher Scientific) following the same protocol as Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR). SupersScript reverse transcriptase (Thermo Fisher Scientific) was used to reverse transcribe nrdA using extension primers 25 and 26 (See Supplementary Table S2). The primer was radioactively labelled with [g- 32 P]ATP using T4 Polynucleotide Kinase (Thermo Fisher Scientific). A DNA fragment of 841 nucleotides corresponding to the sequence upstream of the nrdA coding region was amplified by PCR using High Expand Taq polymerase (Roche). The resulting PCR products generated a sequence ladder with the same primer used for the extension reaction using the Sequenase kit (Promega). The extension product was resolved on an 8% acrylamide – 7M urea gel alongside the sequence ladders. 5’RACE (5’ Rapid alignment of cDNA ends) P. aeruginosa PAO1 cultures were collected under aerobic and anaerobic conditions during the exponential growth phase (OD 600 = 0.5), and RNA extraction was performed as previously described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR). cDNA synthesis was performed using Maxima Reverse Transcriptase (Thermo Fisher Scientific), according to the manufacturer’s instructions. Each reaction contained 1.5 pmol of gene-specific primer (44, Supplementary Table S2) and 0.3 mg of total RNA. The resulting cDNA was purified using the High Pure PCR Product Purification Kit (Roche Life Science), according to the manufacturer’s instructions. Poly-A tailing was performed using 40 U of Terminal Deoxynucleotidyl Transferase (Thermo Fisher Scientific) and 1.5 nmol dATP (Thermo Fisher Scientific). The first PCR (PCR1) was conducted using primers45 and 24 (Supplementary Table S2) with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific), using the poly-A cDNA as a template. PCR1 was optimised with 10 cycles of 40-second extension, followed by 25 cycles with a 20-second extension, increasing by 5 seconds per cycle. The PCR1 product was gel-purified using the GeneJet Gel Extraction kit (Thermo Fisher Scientific) and diluted 1:20. The second PCR (PCR2) was performed using primers 46 and 47 (Supplementary Table S2), with PCR1 as a template, using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) according to the manufacturer’s instructions. PCR2 products were purified and cloned into the pJET1.2 blunt vector, following the same protocol as described in Materials and Methods Section Plasmid construction). Colonies were sequenced by Eurofins Genomics using the primer 47 (Supplementary Table S2). Transcriptional shut-off assay P. aeruginosa PAO1 wild-type , P. aeruginosa Δ5’UTR P. aeruginosa pETS134, and P. aeruginosa pETS257 strains were grown and treated with 200 µg/ml of rifampicin (a transcriptional blocking agent). Samples were collected at 0, 5, and 20 minutes after rifampicin addition to arrest transcription for P. aeruginosa pETS134 and pETS257, and at 0, 2, 6, 10, and 20 minutes for P. aeruginosa PAO1 wild-type , and Δ5’UTR. Total RNA was extracted using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reverse transcription and subsequent quantitative real-time PCR (qRT-PCR) measurements were performed as described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR), with the modification that the 16S rRNA gene was used as an internal control and nrdJ as the reference gene. Absolute PCR quantification was performed using a standard curve from target genes ( cat , nrdA , nrdJ , 16S). Briefly, PCR amplification of genomic nrdA , nrdJ , and 16S rRNA gene, and cat from pETS130-GFP plasmid was performed using the primers listed on Supplementary Table S2. A ten-fold dilution of known amplicons quantities was performed, and the number of copies was calculated by the following formula (Integrated DNA Technologies, IDT): $$\:Number\:of\:copies=\frac{Quantity\:of\:DNA\:\left(ng\right)\times\:6.022\times\:{10}^{23}}{Length\:of\:DNA\:\left(bp\right)\times\:1\times\:{10}^{9}\times\:650}$$ In which \(\:6.022\times\:{10}^{23}\) is Avogadro number, 650 is the average weight (grams, g) of a base pair (bp) in Daltons and \(\:1\times\:{10}^{9}\) is to convert grams(g) to nanograms (ng). Once the Ct values from each dilution of every target gene were determined, a standard curve was established by plotting the target DNA copy number against each Ct value. The standard curve was used to calculate the number of copies from each target gene in different samples. The percentage of mRNA retro-transcribed remaining was calculated using non-rifampicin treated sample or the before-rifampicin sample as the initial mRNA amount of each target gene. Data were analyzed using nonlinear regression analysis in GraphPad Prism 10.1.1 (GraphPad Software). A one-phase decay model was applied assuming a decay from a shared initial value (Y 0 = 100). The equation model was: \(\:Y=\left({Y}_{0}-Plateau\right)\times\:{e}^{-K\times\:X}+Plateau\) , where X is time (minutes) and Y is percentage (%) of remaining cDNA that decays with one phase down to a Plateau, and K is the decay rate constant. To ensure biological plausibility and stability of the fit, constraints were applied: K > 0.01 and in some cases, Plateau values were fixed when instability was detected in the fitting process. Key parameters extracted from the fit included the decay rate constant (K) and half-life ( \(\:{t}_{1/2}=\frac{(\text{ln}2)}{K}\) ). For the sample nrdA transcript sample within WT + P nrdA - Δ5’UTR parameters determination also included fitting the plateau (Plateau = 6) to ensure that the calculated values accurately reflect the true behaviour of the system. Comparisons between the groups focused on differences in K and half-life as indicators of decay rate. The goodness of the fit was evaluated using the R 2 value and 95% confidence intervals (profile likelihood method). In some cases (Δ5’UTR, nrdJ transcript), profile likelihood intervals could not be fully determined and were calculated using the symmetrical (asymptotic) method implemented in GraphPad Prism 10.1.1 (GraphPad Software). While this method assumes normality and may underestimate the uncertainty for some parameters, it provides a consistent basis for comparison. Galleria mellonella infection Galleria mellonella was maintained and dose-infected as described before 21 with some modifications. The different doses were plated in LB (Scharlab) plates to determine bacterial CFU (20–40 CFUs per larva). Regarding bacterial RNA extraction during infection, groups of 10 bacterial-infected larvae were collected about 16-17h after the course of infection and anesthetized on ice for 10 min. Bacterial sample collection from Galleria mellonella was performed following the protocol described before 21 and RNA extraction was performed as described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR). In the case of Galleria mellonella virulence analysis, groups of 10 larvae were infected and kept at 37ºC and periodically monitored during infection. Bioinformatic analysis and data sourcing Riboswitch prediction analysis was performed using Riboswitch Scanner 22 , 23 with default settings. RegPrecise 24 was used to identify cobalamin regulon genes in P. aeruginosa PAO1. Sequences obtained from RegPrecise database and putative B 12 riboswitch sequences were aligned using T-coffee software 25 with default parameters. The alignment was manually analysed, considering the predicted secondary structure. BLAST software 26 and the Coding Potential Calculator 2 (CPC2) tool 27 were used for protein-coding potential prediction, both with default settings. The Rfam 28 and RNAcentral 29 databases were used to submit 5’UTR sequence to identify potential matches with non-coding RNA sequences. The presence of Rho-independent terminators was evaluated using ARNold software 30 with standard settings. Promoter prediction was conducted using PromoterHunter 31 and BPROM 32 . Both scientific publications and data repositories were consulted, including Kyoto Encyclopaedia of genes and genomes 33 , as well as raw data from several RNA-seq experiments, with the specific sources cited in the relevant sections of the manuscript. Statistical analysis Statistical analyses were performed using GraphPad Prism 10.1.1 (GraphPad Software). Single comparisons were performed using unpaired Student’s t -test. Statistical significance was indicated by p -values as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Error bars in figures represent the standard deviation (SD) between samples. Welch's t-test was applied when unequal variances were detected. Conclusions The 5’ UTR appears to regulate nrdAB mRNA transcription and stability and may serve as a molecular mechanism to down-regulate nrdAB expression during the stationary phase, when the synthesis of dNTPs is no longer required for DNA replication. To date, no mechanisms have been described in P. aeruginosa that explain how nrdA transcription is down-regulated upon entry into stationary phase. The absence of the 5’ UTR from the nrdAB class Ia RNR operon in P. aeruginosa PAO1 led to increased transcription, enhanced mRNA stability and greater structural flexibility (Figure. 8). These changes ultimately result in higher NrdA protein levels (Fig. 8 ). This effect is likely due to the increased number of nrdAB mRNA copies and their improved stability, which together enhance ribosome accessibility and translation efficiency, rather than an increase in translation initiation itself. However, instead of conferring an adaptive advantage, the loss of the 5’ UTR appears to disrupt the normal regulatory balance between RNR classes ( nrdA and nrdJ ), leading to reduced virulence of P. aeruginosa PAO1 during Galleria mellonella infection (Fig. 8 ). These findings highlight the crucial role of the 5’UTR in fine-tuning nrdAB operon expression and underscore the importance of untranslated regions as regulatory elements. Nevertheless, further studies are needed to elucidate the molecular mechanisms underlying nrdAB modulation and its integration into broader regulatory networks. Declarations Acknowledgments We thank to Dr. Susanne Häußler and Dr. Alejandro Arce-Rodriguez for kindly providing CRISPR-Cas9 toolkit plasmids and for their technical support. Funding This study was partially supported by grants PID2021-125801OB-100, PLEC2022-009356 and PDC2022-133577-I00, funded by MCIN/AEI/ 10.13039/501100011033 and “ERDF A way of making Europe”, the CERCA programme, and AGAUR-Generalitat de Catalunya (2021SGR01545), the European Regional Development Fund (FEDER), the Catalan Cystic Fibrosis association, and Obra Social “La Caixa”. E.T. is a researcher of the ICREA Academia 2025 program. A.M-M thanks Generalitat de Catalunya for its financial support through the FI program (FI_B00313). Author Contributions Statement A.M-M and ET wrote the manuscript. A.M-M, AR and LP performed the biological assays. ET supervised research, managed the project, secured funding, and reviewed the experimental data. All authors have read and approved the final version of the manuscript. Data Availability Statement All data supporting the findings of this study, including full-length original gels and blots, are available in the Supplementary Material and at the public repository: https://doi.org/10.34810/data2361. Competing Interests Statement The authors declare no competing interests. References Letizia, M., Diggle, S. P. & Whiteley, M. Pseudomonas aeruginosa : ecology, evolution, pathogenesis and antimicrobial susceptibility. Nat Rev Microbiol 1–17 (2025) doi:10.1038/S41579-025-01193-8. Stover, C. K. et al. 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Supplementary Files MartinezMateosASupplementaryMaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviews received at journal 12 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers invited by journal 08 Jul, 2025 Editor assigned by journal 08 Jul, 2025 Editor invited by journal 08 Jul, 2025 Submission checks completed at journal 29 Jun, 2025 First submitted to journal 29 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6937143","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483036489,"identity":"6ab6b545-8511-414d-bd69-7929d22e2711","order_by":0,"name":"Ángela Martínez-Mateos","email":"","orcid":"","institution":"The Barcelona Institute of Science and Technology (BIST)","correspondingAuthor":false,"prefix":"","firstName":"Ángela","middleName":"","lastName":"Martínez-Mateos","suffix":""},{"id":483036491,"identity":"bf9db0af-5023-47fc-b0df-9ac1fbb24dd1","order_by":1,"name":"Alba Rubio-Canalejas","email":"","orcid":"","institution":"The Barcelona Institute of Science and Technology (BIST)","correspondingAuthor":false,"prefix":"","firstName":"Alba","middleName":"","lastName":"Rubio-Canalejas","suffix":""},{"id":483036502,"identity":"9dcc2339-b610-4679-817b-2678b6fb7fc8","order_by":2,"name":"Lucas Pedraz","email":"","orcid":"","institution":"The Barcelona Institute of Science and Technology (BIST)","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"","lastName":"Pedraz","suffix":""},{"id":483036503,"identity":"11ef116e-365e-44ad-b6ba-b58473664801","order_by":3,"name":"Eduard Torrents","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYFCCBCBmQ3DlGCSAJA8RWiRgHGPStSQ2ENLCz55j+LmijKGOX/rsw8eFP+rSN9xuYHzwtg23FsmeN8aSZ84xSEj2pRsbz0g4nLvhzgFmw7l4tBjcyDGQbGxjkDA4w8YmzZNwIHfDjQQ2aV48Wuxv5Bj/hGph/82TUJducCOB/Tc+LQYSOWZwW5h5EpgTgFrYmPFpkTjzrMyy4ZyE5MweNmZpnrTDhjPvHGyWnHMOtxb+9uTNNxvKbPj5edgYP/PY1Mnz3W4++OFNGW4tMMuQOYwNBNWPglEwCkbBKMAPABFrSRg+8ffPAAAAAElFTkSuQmCC","orcid":"","institution":"The Barcelona Institute of Science and Technology (BIST)","correspondingAuthor":true,"prefix":"","firstName":"Eduard","middleName":"","lastName":"Torrents","suffix":""}],"badges":[],"createdAt":"2025-06-20 08:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6937143/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6937143/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-20079-6","type":"published","date":"2025-10-15T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86518297,"identity":"4fa2b78b-e5c7-463c-9c87-0e32ab0b2bd6","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":380627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of th\u003c/strong\u003ee \u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eupstream region\u003c/strong\u003e.\u003cstrong\u003e (a)\u003c/strong\u003e Schematic representation of the \u003cem\u003enrdAB\u003c/em\u003epromoter and its genetic context. The AlgR binding site is indicated in green, and NrdR binding sites are shown in red. The ribosome binding site (RBS), translational initiation codon (Met), and the -35 and -10 promoter regions are underlined. Transcriptional start sites (TSS) identified by different methods are marked with coloured triangles, as detailed in panel B. The long untranslated region (5’ UTR) of \u003cem\u003enrdA \u003c/em\u003e(408-411 bp) is highlighted in light orange (\u003cstrong\u003eb)\u003c/strong\u003e Identification of TSSs using various approaches, including primer extension and 5’rapid amplification of cDNA ends (5’ RACE), both carried out in our laboratory, as well as transcriptomic data from differential RNA-seq (dRNA-seq) experiment. Coloured triangles correspond to the TSS identified by each method. (\u003cstrong\u003ec) \u003c/strong\u003ePrimer extension analysis showing the identified TSS (blue triangle in Figure 1a-b), indicated with an asterisk (*). \u003cstrong\u003e(d)\u003c/strong\u003e 5’RACE analysis of the \u003cem\u003enrdA\u003c/em\u003epromoter, with the identified TSS (pink triangle, Figure 1a-b). The percentage indicates the proportion of sequenced colonies that matched the same region, with the number matching the exact base pair shown in parentheses. Original and cropped version of the gel is provided in the Supplementary Material S4 and is also available in the public repository (\u003ca href=\"https://doi.org/10.34810/data2361\"\u003ehttps://doi.org/10.34810/data2361\u003c/a\u003e)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/494f31ec3121930371c4c1a3.png"},{"id":86519614,"identity":"f445f932-bd48-44f1-b6db-f9cc68a4abdf","added_by":"auto","created_at":"2025-07-11 14:39:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":672870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCobalamin riboswitch analysis. (a)\u003c/strong\u003e Sequence alignment of representative cobalamin riboswitch in \u003cem\u003eP. aeruginosa\u003c/em\u003e: PA2911 (probable TonB-dependent receptor, \u003cem\u003ebtuB\u003c/em\u003e), PA1271 (Probable TonB-dependent receptor, \u003cem\u003ebtuB2\u003c/em\u003e), PA2946 (hypothetical protein, \u003cem\u003ecbtA\u003c/em\u003e), PA2906 (probable oxidoreductase, \u003cem\u003ecobG\u003c/em\u003e) and PA2945 (conserved hypothetical protein, \u003cem\u003ecobW\u003c/em\u003e) and PA1156 (catalytic component of ribonucleotide reductase class Ia, \u003cem\u003enrdA). \u003c/em\u003ePositions are numbered from the translational start codon (ATG). Predicted secondary structure elements, including loops and hairpins, are annotated based on structural alignments from\u003csup\u003e41\u003c/sup\u003e. Sequence conservation across signature helices (0 to 6) is colour encoded. The underlined region corresponds to the sequence analysed in Figure 3a.\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(b, c) \u003c/strong\u003eTranscriptional and translational \u003cem\u003egfp\u003c/em\u003e reporter assays of \u003cem\u003enrdA\u003c/em\u003e with and without the 5’ UTR in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 WT using pETS130-derived constructs. Assays were performed under various concentrations or in the absence of coenzyme B\u003csub\u003e12\u003c/sub\u003e. Relative fluorescence units (RFU), normalized to OD\u003csub\u003e600\u003c/sub\u003e were recorded over a 5-hours period following coenzyme B\u003csub\u003e12\u003c/sub\u003e addition. Symbols in the graph represent different concentrations of coenzyme B\u003csub\u003e12\u003c/sub\u003e: □ 1 μg/ml, ○2.5 μg/ml and △10 μg/ml). Optical density (OD) was measured at 600 nm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/a9eea03979fc1cc3803a4f47.png"},{"id":86519615,"identity":"5fd9fdef-60c9-4a03-bdbd-1c47e331e7b6","added_by":"auto","created_at":"2025-07-11 14:39:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":444661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 5’UTR does not appear to encode a protein or a small non-coding RNA. (a) \u003c/strong\u003eAnalysis using\u003cstrong\u003e \u003c/strong\u003ethe Coding potential calculator (CCP2) and\u003cstrong\u003e \u003c/strong\u003eBLAST tools. A 49-amino acid peptide predicted by CCP2 was analysed via BLASTp, and the full 5’ UTR sequence was analysed using tBLASTn. (\u003cstrong\u003eb) \u003c/strong\u003eSDS-PAGE (16% acrylamide) of crude extracts from \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 WT harbouring pJN105::5’UTR after induction with 0.4% L-arabinose. (\u003cstrong\u003ec) \u003c/strong\u003eBioinformatic, bibliographic, and experimental identification of transcriptional starting sites (TSS) across the \u003cem\u003enrdAB \u003c/em\u003eupstream and downstream regions, Rho-independent terminators predicted using ARNold software, and small RNAs (sRNA) previously detected\u003csup\u003e47\u003c/sup\u003e. Original and cropped version of the gel is provided in the Supplementary Material S4 and is also available in the public repository (\u003ca href=\"https://doi.org/10.34810/data2361\"\u003ehttps://doi.org/10.34810/data2361\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/565765acda36f2aaddf0d921.png"},{"id":86518304,"identity":"adfb96f7-d535-4fa7-b965-80fe074d5e2e","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":366797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression is increased in the absence of its 5’ UTR during both aerobic and anaerobic growth.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e \u003cem\u003enrdA\u003c/em\u003e expression by measuring RFU/OD\u003csub\u003e600\u003c/sub\u003e values from \u003cem\u003egfp\u003c/em\u003e transcriptional reporter assay with and without the 5’UTR, under aerobic and anaerobic conditions, during exponential and stationary growth phases using pETS130-GFP derivatives.\u003cstrong\u003e (b)\u003c/strong\u003e Fold-change values of \u003cem\u003enrdA\u003c/em\u003e transcript levels measured by RT-qPCR in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 Δ5’UTR (\u003cem\u003enrdA\u003c/em\u003e, chromosomically deleted) compared to the wild-type strain, under aerobic and anaerobic conditions during both exponential and stationary growth phases. (\u003cstrong\u003ec) \u003c/strong\u003eFold-change \u003cem\u003enrdA\u003c/em\u003e RT-qPCR values in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 wild-type and its isogenic Δ5’UTR (\u003cem\u003enrdA\u003c/em\u003e, chromosomically deleted) compared across all growth conditions (aerobic vs. anaerobic, EX vs ST). (\u003cstrong\u003ed) \u003c/strong\u003eSimplified model of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003enrdA\u003c/em\u003e expression dynamics across bacterial growth phases, based on results from transcriptional reporter assays and qRT-PCR analysis. RFU=relative fluorescent units. Significance: \u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001(****).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/20bdaa668166f8ffccef57d6.png"},{"id":86518299,"identity":"9950eac5-41ca-48f4-885d-9f968bd9cdb8","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":307537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional shut-off assay and mRNA half-life and decay rate determination of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enrdJ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003ea)\u003c/strong\u003e retrotranscribed mRNA decay curves and percentage of remaining transcripts of \u003cem\u003enrdA \u003c/em\u003e(with and without 5’ UTR) and \u003cem\u003enrdJ \u003c/em\u003ein \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 wild-type (WT) carrying pETS130-GFP derivatives (WT + pETS134 (WT + P\u003cem\u003enrdA\u003c/em\u003e) and WT + pETS257 (WT + P\u003cem\u003enrdA\u003c/em\u003e- Δ5’UTR)) after rifampicin treatment at 0, 5, and 20 min. (\u003cstrong\u003eb)\u003c/strong\u003e Similar decay analysis in \u003cem\u003eP. aeruginosa \u003c/em\u003ewild-type and PAO1 Δ5’UTR (\u003cem\u003enrdA\u003c/em\u003e) at 0, 2-, 6-, 10- and 20-min post-rifampicin treatment. (\u003cstrong\u003ec, d\u003c/strong\u003e) One-phase exponential decay model and best-fit mRNA half-life values (minutes) and constant decay rate (k) in minutes\u003csup\u003e-1\u003c/sup\u003e for each transcript, normalized to the endogenous control 16S rRNA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/25b63fad80049057123e0593.png"},{"id":86518301,"identity":"f8b00be8-93b2-49a8-9e19-30445b313fd9","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe absence of the 5’UTR results in a more flexible RNA structure, promoting \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranslation. (a) \u003c/strong\u003eTable of minimum free energy values for predicted secondary structures of \u003cem\u003enrdAB\u003c/em\u003e mRNA with and without 5’UTR, and free energy values for RBS (Shine Dalgarno sequence) binding, calculated using RNAfold software. (\u003cstrong\u003eb) \u003c/strong\u003eMountain plots of predicted optimal secondary structures, with the x-axis representing the cumulative number of base pairs up to that position and the y-axis the localization of each bp. Blue and green arrows denote the 5’ and 3’ ends of the mRNAs, respectively. (\u003cstrong\u003ec) \u003c/strong\u003eWestern blot analysis of NrdA protein expression in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 WT and PAO1 Δ5’UTR (\u003cem\u003enrdA\u003c/em\u003e) strains during exponential and stationary phases under both aerobic and anaerobic conditions. Original and cropped version of the gel is provided in the Supplementary Material S4 and is also available in the public repository (\u003ca href=\"https://doi.org/10.34810/data2361\"\u003ehttps://doi.org/10.34810/data2361\u003c/a\u003e)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/6847542721495abac0e13e57.png"},{"id":86518306,"identity":"dfffdb58-8260-4418-9553-ea1cd63d23bd","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":136081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVirulence analysis during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGalleria mellonella \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection. (a) \u003c/strong\u003eKaplan-Meier survival analysis of \u003cem\u003eGalleria mellonella \u003c/em\u003elarvae infected with \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 WT and PAO1 Δ5’UTR\u003cstrong\u003e \u003c/strong\u003e(\u003cem\u003enrdA\u003c/em\u003e).\u003cstrong\u003e \u003c/strong\u003ePBS-injected larvae served as controls. Larval survival was monitored for 15-20 h post-injection, with observations recorded at 15, 16, 17, 18, 19 and 20 h\u003cstrong\u003e. (b) \u003c/strong\u003eMedian survival time (MST) of \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 WT and PAO1 Δ5’UTR infected larvae. \u003cstrong\u003e(c) \u003c/strong\u003eFold-change \u003cem\u003enrdA\u003c/em\u003eRT-qPCR values in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 wild-type and its isogenic Δ5’UTR (\u003cem\u003enrdA\u003c/em\u003e, chromosomically deleted) infected larvae.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/d7fe8932a9a75f3c2d824e9e.png"},{"id":86518309,"identity":"df6e5266-6b70-48ef-9462-d44138d3bb05","added_by":"auto","created_at":"2025-07-11 14:31:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":309080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summary comparing the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wild-type form (left) and the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enrdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eΔ5’UTR mutant (right). \u003c/strong\u003eHighlighting the main regulatory and phenotypic effects on \u003cem\u003enrdA\u003c/em\u003e expression (top), effects on ribonucleotide reductase (RNR) homeostasis (middle) and virulence in \u003cem\u003eGalleria mellonella\u003c/em\u003e (bottom). Created with BioRender.com\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/1e79d98cb332c67cd789ef55.png"},{"id":93955954,"identity":"67897310-e04b-4e67-abaa-d8ecef3008d5","added_by":"auto","created_at":"2025-10-20 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3998971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/8fb35734-f7fa-4299-85e6-38a335faaaa9.pdf"},{"id":86520088,"identity":"d0f94ac8-d699-4b90-a791-c7983631e78f","added_by":"auto","created_at":"2025-07-11 14:47:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":977533,"visible":true,"origin":"","legend":"","description":"","filename":"MartinezMateosASupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6937143/v1/a787906d140dcc5b6a7d029f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The long 5′UTR of nrdAB modulates transcription, mRNA stability, and virulence in Pseudomonas aeruginosa","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a highly adaptable opportunistic pathogen that exhibits a notable metabolic flexibility, genomic variability and phenotypic diversity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Due to its ability to survive in diverse environments \u0026mdash;along with its both acquired and innate antibiotic resistance mechanisms\u0026mdash; and its capability to form biofilms, there is an urgent need for the discovery of new therapeutic strategies\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOne promising therapeutic target is ribonucleotide reductase (RNR), a family of metalloenzymes responsible for the \u003cem\u003ede novo\u003c/em\u003e synthesis of deoxyribonucleotides (dNTPs) through a radical-based chemistry mechanism\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Three main classes of RNRs have been described: class I (subtypes Ia, Ib, Ic, Id and Ie)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, Class II and Class III\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This classification is based on structural differences, oxygen needs, metallocofactor requirements and the mechanism used for their radical generation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eP. aeruginosa\u003c/em\u003e is one of the few organisms known to encode all three main classes, reflecting the finely tuned regulatory systems of this microorganism\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough class I RNRs are among the best-characterized enzymes, little is known about class Ia in \u003cem\u003eP. aeruginosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This RNR class consists of two homodimeric subunits (α and β), encoded by the \u003cem\u003enrdAB\u003c/em\u003e operon. So far, two transcriptional factors (AlgR and NrdR) have been identified as regulators of the operon\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, key aspects of the regulatory networks of \u003cem\u003enrdAB\u003c/em\u003e operon remain unclear. Several years ago, a 5\u0026rsquo; untranslated region (5\u0026rsquo;UTR) upstream \u003cem\u003enrdA\u003c/em\u003e was predicted through bioinformatic analysis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, but further experimental validation is needed to confirm its structure and regulatory role.\u003c/p\u003e\u003cp\u003eThe 5\u0026rsquo;UTRs of bacterial mRNAs have increasingly been recognized as crucial regulatory elements that influence gene expression at multiple levels. Rather than being passive sequences between promoters and start codons, 5\u0026rsquo;UTRs harbor a wide variety of regulatory elements that actively control translation efficiency, transcript stability and transcription termination\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Their functional diversity includes ribosome binding sites (RBS) or Shine-Dalgarno (SD) sequences, upstream open reading frames (uORFs), binding motifs for small RNAs (sRNAs), and dynamic secondary structures capable of responding to environmental or metabolic signals, among others\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eChanges in 5\u0026rsquo;UTR sequence or structure can alter gene expression by affecting ribosome accessibility, modulating mRNA degradation, or engaging in complex networks with trans-acting elements like sRNAs and RNA-binding proteins\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For example, riboswitches within 5\u0026rsquo;UTRs bind metabolites, ions, or coenzymes to control downstream gene expression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven that transcription and translation are tightly coupled in bacteria and they occur in the cytoplasm, the regulatory information embedded in 5\u0026rsquo;UTRs plays a particularly prominent role in post-transcriptional control\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Notably, 5\u0026rsquo;UTRs do not function independently, as recent studies suggest that riboswitches, sRNAs, antisense RNAs, and 5\u0026rsquo;UTRs themselves can regulate one another, forming intricate regulatory networks\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite their importance, many 5\u0026rsquo;UTRs remain poorly characterized, and their potential as tunable regulatory modules in bacterial gene expression is only beginning to be uncovered\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor this reason, the aim of this study is to understand the nature and functional role of the predicted 5\u0026rsquo;UTR of \u003cem\u003enrdAB\u003c/em\u003e operon in \u003cem\u003eP. aeruginosa.\u003c/em\u003e By exploring its potential impact on the regulation of \u003cem\u003enrdAB\u003c/em\u003e, we seek to determine whether it influences transcript stability, translation efficiency or gene expression dynamics under different conditions.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eThe\u003c/b\u003e \u003cb\u003enrdAB\u003c/b\u003e \u003cb\u003eoperon encodes a long 5\u0026rsquo; untranslated region (5\u0026rsquo;UTR) with an unknown function.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe oxygen-dependent class Ia ribonucleotide reductase (RNR) of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is encoded by two cotranscribed genes, \u003cem\u003enrdA\u003c/em\u003e and \u003cem\u003enrdB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In a previous study carried out by our group, several regulatory features of the \u003cem\u003enrdAB\u003c/em\u003e operon were identified. On one hand, transcriptional binding sites for AlgR and NrdR were identified\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). On the other hand, bioinformatic analyses revealed that the transcription starting site (TSS) of \u003cem\u003enrdAB\u003c/em\u003e was located approximately 400 bp upstream of the translational start codon (ATG), indicating the presence of a long 5\u0026rsquo;UTR\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, yellow triangle). Based on this information, the putative \u0026minus;\u0026thinsp;10 and \u0026minus;\u0026thinsp;35 promoter boxes of the \u003cem\u003enrdAB\u003c/em\u003e operon were mapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo experimentally validate the transcription start site of the \u003cem\u003enrdAB\u003c/em\u003e mRNA, we combined insights from laboratory experiments and genome annotation databases, which consistently indicated that the TSS is located between 408 and 411 bp upstream of the translational start site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b). A primer extension assay confirmed that the 5\u0026rsquo; end of the \u003cem\u003enrdAB\u003c/em\u003e transcript begins with a cytosine nucleotide located 411 bp upstream of the predicted translational start site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c, blue triangle), while 5\u0026rsquo;RACE positioned the TSS 409 bp upstream (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, d, pink triangle) corroborating the presence of a long 5\u0026rsquo;UTR.\u003c/p\u003e\u003cp\u003eGiven that bacterial 5\u0026rsquo; UTR are typically short (20\u0026ndash;30 nucleotides), the presence of such an extended 5\u0026rsquo;UTRs suggests a potential regulatory element involved in modulating \u003cem\u003enrdA\u003c/em\u003e expression\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo;UTR does not appear to contain a cobalamin riboswitch.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLong 5\u0026rsquo;UTRs have been associated with several regulatory elements\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Among these are riboswitches, the vast majority of which are located in the 5\u0026rsquo;UTRs of mRNA\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. A bioinformatic analysis using Riboswitch Scanner identified a potential cobalamin riboswitch within its 5\u0026rsquo;UTR (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Notably, the \u003cem\u003enrdABS\u003c/em\u003e operon, which encodes the class Ia RNR in \u003cem\u003eStreptomyces\u003c/em\u003e, is regulated by a cobalamin riboswitch\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This is not an isolated case, several RNRs, including the class Ia enzyme from \u003cem\u003eBacillus halodurans\u003c/em\u003e, are also regulated by B\u003csub\u003e12\u003c/sub\u003e riboswitches\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven this context, it was reasonable to investigate the presence of a similar regulatory element in the 5\u0026rsquo;UTR of the \u003cem\u003eP. aeruginosa nrdA\u003c/em\u003e gene. Furthermore, a known connection exists between vitamin B\u003csub\u003e12\u003c/sub\u003e and RNR activity, as demonstrated by the class II RNR from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, which, like other class II RNRs, relies on the availability of S-adenosylcobalamin (vitamin B\u003csub\u003e12\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCobalamin riboswitches constitute a diverse family characterized by a highly conserved secondary structure and a sequence signature known as the B\u003csub\u003e12\u003c/sub\u003e box\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To investigate the presence of a putative B\u003csub\u003e12\u003c/sub\u003e box, a multiple sequence alignment was performed between the predicted \u003cem\u003eP. aeruginosa nrdA\u003c/em\u003e cobalamin riboswitch and various \u003cem\u003eP. aeruginosa\u003c/em\u003e cobalamin riboswitches obtained from the RegPrecise database\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (See Materials and Methods). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the alignment revealed a conserved putative B\u003csub\u003e12\u003c/sub\u003e box and structural similarities between the predicted sequence alignment from\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and the \u003cem\u003enrdA\u003c/em\u003e sequence (PA1156) from \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate the presence of a functional B\u003csub\u003e12\u003c/sub\u003e riboswitch, transcriptional and translational GFP fusions of \u003cem\u003enrdA\u003c/em\u003e were constructed (see Materials and Methods), and GFP reporter assays were performed (see Materials and Methods). These fusions served as B\u003csub\u003e12\u003c/sub\u003e-sensors to evaluate the presence of a functional riboswitch and the effect of coenzyme B\u003csub\u003e12\u003c/sub\u003e on downstream gene expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c, the assays did not demonstrate a significant difference in GFP expression across increasing concentrations of coenzyme B\u003csub\u003e12\u003c/sub\u003e in either the transcriptional (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) or translational (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) fusions.\u003c/p\u003e\u003cp\u003eWhile the precise mechanisms of coenzyme B\u003csub\u003e12\u003c/sub\u003e binding and downstream regulation remain poorly understood\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, translational regulation appears to be the most prevalent mechanism among cobalamin riboswitch in γ-Proteobacteria\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been shown that that cobalamin riboswitches are a complex family capable of sensing and responding to a range of corrinoid compounds\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. However, some cobalamin riboswitches are known to respond only to specific subsets of these compounds.\u003c/p\u003e\u003cp\u003eIn addition, the sequence and structure of the \u003cem\u003enrdA\u003c/em\u003e promoter sequence did not perfectly align with those of experimentally validated B\u003csub\u003e12\u003c/sub\u003e riboswitches (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eIn conclusion, although bioinformatic analysis initially predicted the presence of a cobalamin riboswitch in the \u003cem\u003enrdAB\u003c/em\u003e 5' UTR, our experimental results do not support a functional role for coenzyme B\u003csub\u003e12\u003c/sub\u003e in regulating \u003cem\u003enrdAB\u003c/em\u003e expression. Therefore, we cannot conclude that the 5\u0026rsquo;UTR of \u003cem\u003enrdAB\u003c/em\u003e functions as a cobalamin riboswitch.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo;UTR does not encode an upstream open reading frame (uORF) or a small RNA (sRNA).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOther regulatory elements commonly found within 5\u0026rsquo;UTR include upstream ORFs (uORFs), which are short open coding sequences often involved in post-transcriptional regulation through ribosome-mediated translational attenuation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo assess the non-coding nature of the \u003cem\u003enrdAB\u003c/em\u003e 5' UTR, both bioinformatic and experimental approaches were employed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the full 411 bp 5\u0026rsquo;UTR sequence was analysed using the Coding Potential Calculator 2 (CPC2) tool\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, which predicted a 49 amino acid peptide. However, the coding probability was low, and a BLASTp search against the non-redundant protein database (nr) revealed no significant similarity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Similarly, a translated BLAST (tBlastn) search against the core nucleotide database (core nt) yielded less than 30% coverage to hypothetical proteins from various \u003cem\u003eP. aeruginosa\u003c/em\u003e strains\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the untranslated nature of the 5\u0026rsquo;UTR, the entire sequence was cloned downstream of an arabinose-inducible promoter plasmid (See Materials and Methods). Theoretically, the 5\u0026rsquo; UTR could encode a protein of approximately 15 kDa; in parallel, the predicted 49-amino acid peptide would correspond to a protein of less than 10 kDa. However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, no protein bands of either size (10 or 15 kDa) were detected upon arabinose induction, and protein expression profiles under induced and non-induced conditions were identical. These results suggest that the 5\u0026rsquo; UTR does not encode a functional uORFs.\u003c/p\u003e\u003cp\u003eAnother class of regulatory elements frequently found in 5\u0026rsquo; UTR are small RNAs (sRNAs), which are non-coding RNAs that can influence transcription, translation, mRNA stability, and DNA maintenance or silencing\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Most sRNAs are located in intergenic regions (IgRs), transcribed from their own promoter or nearby gene promoters, and often terminate via Rho-independent mechanisms\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To explore this possibility, we analysed the regions upstream and downstream of \u003cem\u003enrdA\u003c/em\u003e for additional TSSs, annotated sRNAs, and Rho-independent terminators.\u003c/p\u003e\u003cp\u003eThe complete 5\u0026rsquo; UTR sequence was submitted to the Rfam\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and RNAcentral\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e database. No annotated non-coding RNA matching this sequence was identified. Promoter prediction tools such as PromoterHunter\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and BPROM\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e revealed two possible promoter regions: one previously identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, purple underlined sequences; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, P1 black arrow), and a second located 226 bp upstream of the \u003cem\u003enrdA\u003c/em\u003e start codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, P2 grey arrow).\u003c/p\u003e\u003cp\u003eAnalysis using ARNold software\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e identified only the known Rho-independent terminator at the end of the \u003cem\u003enrdAB\u003c/em\u003e operon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Additionally, it has been reported a putative sRNA, pant125, on the negative strand between coordinates 1254329\u0026ndash;1254720, overlapping the \u003cem\u003enrdAB\u003c/em\u003e promoter region\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). However, their RNA-seq analysis, which assumed all non-coding regions correspond to intergenic regions, may have underestimated the functional relevance of 5\u0026rsquo;UTRs.\u003c/p\u003e\u003cp\u003eSince no additional TSSs or terminators were found near the pant125 region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), one possible explanation is that pant125 is a product of \u003cem\u003enrdA\u003c/em\u003e transcript processing by RNases, resulting in two transcripts: \u003cem\u003enrdA\u003c/em\u003e mRNA and the putative sRNA. In \u003cem\u003eP. aeruginosa\u003c/em\u003e, RNA degradation and processing are mediated by several enzymes, including RNase E (\u003cem\u003erne\u003c/em\u003e, PA2976), ATP-dependent RNA helicase RhlB (\u003cem\u003erhl\u003c/em\u003e, PA3861) and RNAse R (\u003cem\u003ernr\u003c/em\u003e, PA4937), among others (KEGG pathway: pae03018)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To determine whether one of these RNA processing enzymes might be responsible for processing the \u003cem\u003enrdA\u003c/em\u003e transcript, we conducted GFP transcriptional reporter assays comparing \u003cem\u003enrdA\u003c/em\u003e expression (with and without 5\u0026rsquo;UTR region) in \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 wild-type and RNA processing enzymes mutant strains (Supplementary Figure S2). However, none of the RNA processing enzymes mutants showed increased \u003cem\u003enrdA\u003c/em\u003e expression compared to the wild-type strain (Supplementary Figure S2a). Furthermore, when the \u003cem\u003enrdA\u003c/em\u003e promoter (P\u003cem\u003enrdA\u003c/em\u003e) lacking the 5\u0026rsquo; UTR was tested, differences in expression between strains persisted (Supplementary Figure S3b).\u003c/p\u003e\u003cp\u003eTheoretically, if a specific RNA processing enzymes cleaved \u003cem\u003enrdA\u003c/em\u003e mRNA via 5\u0026rsquo;UTR-dependent mechanisms, such differences should disappear in the absence of the 5\u0026rsquo; UTR. The fact that they did not, combined with the pleiotropic effects of the RNase mutations, complicates the identification of a specific regulatory mechanism.\u003c/p\u003e\u003cp\u003eIn conclusion, the combined bioinformatic and experimental evidence suggests that the \u003cem\u003enrdA\u003c/em\u003e 5\u0026rsquo; UTR does not encode a functional uORF or a regulatory sRNA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo;UTR is involved in\u003c/b\u003e \u003cb\u003enrdA\u003c/b\u003e \u003cb\u003eregulation by enhancing its transcription.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs mentioned earlier, although known transcriptional regulators of \u003cem\u003enrdAB\u003c/em\u003e, such as AlgR and NrdR, have been described\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, important gaps remain in our understanding of both the transcriptional and post-transcriptional regulation of \u003cem\u003enrdAB\u003c/em\u003e, particularly concerning the role of the 5\u0026rsquo;UTR.\u003c/p\u003e\u003cp\u003eThe significance of the 5\u0026rsquo; UTR in \u003cem\u003enrdAB\u003c/em\u003e expression was initially assessed through \u003cem\u003enrdA\u003c/em\u003e transcriptional fusions of \u003cem\u003ePnrdA\u003c/em\u003e with GFP, either including or excluding the 5\u0026rsquo;UTR (See Materials and Methods). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, under aerobic conditions, the presence of the 5\u0026rsquo;UTR (P\u003cem\u003enrdA\u003c/em\u003e) resulted in reduced \u003cem\u003enrdA\u003c/em\u003e expression compared to the construct lacking the 5\u0026rsquo;UTR (P\u003cem\u003enrdA\u003c/em\u003e-∆5\u0026rsquo;UTR), which showed significantly higher expression. Interestingly, under anaerobic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), even though class Ia is enzymatically active primarily under aerobic conditions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e the 5\u0026rsquo; UTR exerted a similar repressive effect on \u003cem\u003enrdA\u003c/em\u003e expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, transcriptional fusion experiments revealed growth-phase-dependent differences in expression, also influenced by the presence of the 5\u0026rsquo; UTR. For constructs containing the 5\u0026rsquo;UTR (P\u003cem\u003enrdA\u003c/em\u003e), expression decreased during the stationary phase compared to the exponential phase under both aerobic and anaerobic conditions. In contrast, the P\u003cem\u003enrdA\u003c/em\u003e-∆5\u0026rsquo;UTR fusion showed increased expression during the aerobic stationary phase relative to exponential growth. However, this increase was not observed under anaerobic conditions, where expression decreased during the stationary phase, mirroring the pattern seen with the P\u003cem\u003enrdA\u003c/em\u003e construct.\u003c/p\u003e\u003cp\u003eTo validate these findings in a more physiological context, \u003cem\u003enrdA\u003c/em\u003e expression was compared between \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 wild-type and a specific chromosomal deletion mutant generated using the CRISPR-cas9 system\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, lacking the \u003cem\u003enrdA\u003c/em\u003e 5\u0026rsquo;UTR (PAO1 ∆5\u0026rsquo;UTR) (See Materials and Methods). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cem\u003enrdA\u003c/em\u003e expression was consistently higher in the ∆5\u0026rsquo;UTR mutant compared to the wild-type strain, under both aerobic and, especially, anaerobic conditions, corroborating the results obtained with the GFP reporter assay.\u003c/p\u003e\u003cp\u003eAdditionally, expression dynamics across growth phases reflected those observed with the plasmid-based reporter system: in PAO1 WT, \u003cem\u003enrdA\u003c/em\u003e expression was higher during the exponential phase than in the stationary phase under both aerobic and anaerobic conditions. However, in PAO1 ∆5\u0026rsquo;UTR, \u003cem\u003enrdA\u003c/em\u003e expression remained relatively stable across exponential and stationary growth phases, regardless of oxygen availability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eAs simplified model is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed. Under aerobic conditions, both approaches, GFP transcriptional fusion and qRT-PCR, were consistent: \u003cem\u003enrdA\u003c/em\u003e expression was higher in the absence of the 5\u0026rsquo;UTR and increased progressively throughout bacterial growth phases. Under anaerobic conditions, although \u003cem\u003enrdA\u003c/em\u003e expression without 5\u0026rsquo; UTR was also higher during the stationary phase, a discrepancy emerged between the two methods. In the GFP transcriptional assay, expression appeared to decrease in the stationary phase, whereas qRT-PCR quantifies mRNA levels directly, showed increased expression. This difference may be attributed to the nature of the assays, the plasmid-based reporter system measures expression indirectly, while the chromosomal mutant reflects a more physiologically relevant context. Thus, the latter is considered more reliable.\u003c/p\u003e\u003cp\u003eIn summary, these findings highlight the regulatory complexity of class Ia RNR in \u003cem\u003eP. aeruginosa\u003c/em\u003e, which is influenced by both growth phase and oxygen availability. The data suggest that the 5\u0026rsquo;UTR of \u003cem\u003enrdA\u003c/em\u003e plays a key role in downregulating transcription and reducing RNR protein expression and enzymatic activity during growth, particularly in stationary phases when dNTPs synthesis is no longer required due to the cessation of DNA replication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo;UTR influences\u003c/b\u003e \u003cb\u003enrdAB\u003c/b\u003e \u003cb\u003estability and decay.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the absence of the 5\u0026rsquo;UTR appears to increase \u003cem\u003enrdA\u003c/em\u003e transcription and, consequently, translation. 5\u0026rsquo;UTRs are known to harbour multiple regulatory elements that can contribute to post-transcriptional regulation of the \u003cem\u003enrdAB\u003c/em\u003e operon\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOne such regulatory mechanism involves mRNA stability and decay. To investigate the mRNA half-life of \u003cem\u003enrdAB\u003c/em\u003e, we employed two experimental approaches using the same methodology (See Materials and Methods). In the first approach, we indirectly estimated \u003cem\u003enrdA\u003c/em\u003e half-life using a plasmid-based system, where the \u003cem\u003ecat\u003c/em\u003e gene (chloramphenicol acetyltransferase) served as a reporter for \u003cem\u003enrdA\u003c/em\u003e expression. mRNA decay was analysed using first-order kinetics (See Materials and Methods). In the second approach, we measured \u003cem\u003enrdA\u003c/em\u003e half-life from the chromosomal copy of \u003cem\u003enrdAB\u003c/em\u003e in both \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 wild-type and PAO1 Δ5\u0026rsquo;UTR strains (See Materials and Methods).\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the degradation rate (K) of \u003cem\u003enrdA\u003c/em\u003e transcript lacking the 5\u0026rsquo;UTR (WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR) was slower, with a best fit value of 0.07 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(95% CI: 0.03768 to 0.1352 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the wild-type construct (WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e) which has a 0.21 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e decay rate (95% CI: 0.1876 to 0.2329). Specifically, best-fit half-life values were 3.32 min (95% CI: 2,976 to 3,695 min) for wild-type \u003cem\u003enrdA\u003c/em\u003e and 9.53 minutes (95% CI: 5.129 to 18.39 min) for \u003cem\u003enrdA\u003c/em\u003e without the 5\u0026rsquo;UTR, indicating that the latter is approximately 3 times more stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, chromosomal \u003cem\u003enrdA\u003c/em\u003e measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) revealed a comparable trend. In the \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR, \u003cem\u003enrdA\u003c/em\u003e mRNA exhibited a reduced decay rate of 0.83 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (95% CI: 0.7624 to 0.8988 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the wild-type which has a decay rate of 1.5 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(95% CI: 1.404 to 1.603 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The best-fit half-life values were 0.46 min for the wild-type (95% CI: 0.4323 to 0.4938 min) and 0.84 min for the Δ5\u0026rsquo;UTR (95% CI: 0.7712 to 0,9091 min), suggesting approximately a 2-fold increase in stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and c).\u003c/p\u003e\u003cp\u003eTo confirm the specificity of this effect, we analysed the expression of \u003cem\u003enrdJ\u003c/em\u003e (the class II ribonucleotide reductase from PAO1) as a reference using both approaches. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the decay curves for \u003cem\u003enrdJ\u003c/em\u003e were nearly identical across all strains and constructs, and the best-fit half-life values were similarly consistent between strains using both approaches. In the plasmidic one-phase decay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) the decay rates (K) were 0.44 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (95% CI: 0.4361 to 0.4499 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for the WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e construct compared to 0.30 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (95% CI: 0.2618 to 0.3158 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for the WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR construct. A similar trend was observed the chromosomal one-phase decay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), where the decay rates were 1.96 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (95% CI: 1.818 to 2.149 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for WT and 2.1 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Δ5\u0026rsquo;UTR (95% CI: 1.263 to 2.913 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Half-life values followed the same pattern: in the plasmidic context (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), 1.57 min (95% CI: 1.541 to 1.589 min) for WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e construct compared to 2.42 min (95% CI: 2.195 to 2.648 min) for WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR; and in the chromosomal context (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), 0.35 min (95% CI: 0.3225 to 0.3813 min) for WT compared to 0.33 min (95% CI: 0.2379 to 0.5490 min) for Δ5\u0026rsquo;UTR.\u003c/p\u003e\u003cp\u003eDifferences between the plasmidic and the chromosomal experiments consistently demonstrate that the 5\u0026rsquo; UTR specifically influences the half-life and decay of \u003cem\u003enrdA\u003c/em\u003e but does not affect \u003cem\u003enrdJ\u003c/em\u003e. Despite the methodological variations and some statistical limitations in certain cases, the overall trend is maintained across both experimental systems, corroborating the same conclusion regarding the role of the 5\u0026rsquo; UTR in \u003cem\u003enrdA\u003c/em\u003e stability. Additionally, bacterial gene expression is influenced not only by the 5\u0026rsquo; UTR but also by the coding sequence and downstream reporter gene\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo;UTR-lacking\u003c/b\u003e \u003cb\u003enrdA\u003c/b\u003e \u003cb\u003etranscript appears more structurally flexible, promoting translation.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe core regulatory function of 5\u0026rsquo;UTRs involves their dynamic secondary structures, which can influence translation attenuation, transcript stability, and ribosome accessibility\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur findings suggest that the \u003cem\u003enrdAB\u003c/em\u003e 5\u0026rsquo;UTR acts as a regulatory element that balances \u003cem\u003enrdA\u003c/em\u003e expression, as its absence results in increased transcription and translation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This may be attributed to increased mRNA levels, and consequently more protein, as well as slightly increased stability of the ∆5\u0026rsquo;UTR mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, we sought to determine whether this effect is also driven by structural differences that promote translation.\u003c/p\u003e\u003cp\u003eTo address this, we used RNAfold (See Materials and Methods) to predict the secondary structures of \u003cem\u003enrdAB\u003c/em\u003e transcripts with and without the 5\u0026rsquo; UTR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, both transcripts exhibit nearly identical minimum free energy values (-1950.10 Kcal/mol for \u003cem\u003enrdAB\u003c/em\u003e and \u0026minus;\u0026thinsp;1819.6 Kcal/mol for \u003cem\u003enrdAB\u003c/em\u003e without 5\u0026rsquo;UTR). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb presents mountain plots of their optimal predicted structures, with arrows indicating the 5\u0026rsquo; and 3\u0026rsquo; ends. While the central regions of both transcripts are similar, the 5\u0026rsquo; and 3\u0026rsquo; ends of the wild-type transcript appear to be more structured and complex compared the ∆5\u0026rsquo;UTR variant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWestern-blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) confirmed that NrdA protein levels are elevated in the Δ5\u0026rsquo;UTR strain under both aerobic and anaerobic conditions, during both exponential and stationary growth phases, consistent with the transcriptional data (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This supports the hypothesis that the more flexible RNA structure of the ∆5\u0026rsquo;UTR transcript enhances ribosome access and translation efficiency.\u003c/p\u003e\u003cp\u003eHowever, RNAfold predictions showed no significant difference in total free energy of binding at the ribosome binding site (RBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, -11.46 Kcal/mol for \u003cem\u003enrdAB\u003c/em\u003e and \u0026minus;\u0026thinsp;11.44 Kcal/mol for \u003cem\u003enrdAB\u003c/em\u003e without 5\u0026rsquo;UTR), implying similar RBS accessibility.\u003c/p\u003e\u003cp\u003eTherefore, the data suggest that the 5\u0026rsquo;UTR primarily regulates \u003cem\u003enrdA\u003c/em\u003e at the transcriptional level rather than through translational initiation and may be responsible of the down-regulation of \u003cem\u003enrdA\u003c/em\u003e expression during stationary growth, when dNTP synthesis is no longer required.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe 5\u0026rsquo; UTR modulates\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003epathogenicity during\u003c/b\u003e \u003cb\u003eGalleria mellonella\u003c/b\u003e \u003cb\u003einfection.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have shown that \u003cem\u003enrdJ\u003c/em\u003e is strongly upregulated during \u003cem\u003eG. mellonella\u003c/em\u003e infection, whereas \u003cem\u003enrdA\u003c/em\u003e plays a more minor role\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.To assess whether the increased \u003cem\u003enrdA\u003c/em\u003e transcription and NrdA protein levels resulting from 5\u0026rsquo;UTR deletion affects \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 virulence, we performed infection experiments in \u003cem\u003eG. mellonella\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents virulence analysis using the \u003cem\u003eG. mellonella\u003c/em\u003e infection model. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea Kaplan-Meier survival curves are shown for larvae infected with \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 wild-type and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR (\u003cem\u003enrdA\u003c/em\u003e) (chromosomal deletion). The curves were significantly different according to the Log-rank (Mantel-Cox) test (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), showing a higher statistically significant Median Survival Time (MST) of \u003cem\u003eG. mellonella\u003c/em\u003e larvae infected with \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR (22 hours) compared to those infected with PAO1 WT (20 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn parallel, RT-qPCR was performed to analyse the expression of \u003cem\u003enrdA\u003c/em\u003e in both strains during infection. Samples were collected at 16 h post-infection (See Materials and Methods). Fold Change values were higher in \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR compared to PAO1 WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eWe hypothesize that increased \u003cem\u003enrdA\u003c/em\u003e expression in the ∆5\u0026rsquo;UTR strain does not confer a fitness advantage. Instead, it may disrupt the balance between \u003cem\u003enrdA\u003c/em\u003e and \u003cem\u003enrdJ\u003c/em\u003e expression under both aerobic and anaerobic conditions, at both mRNA and protein levels (see Supplementary Figure S3a and S3c) as well as during infection (see Supplementary Figure S3b). Specifically, \u003cem\u003enrdJ\u003c/em\u003e expression is decreased at the mRNA level under aerobic conditions but not under anaerobic conditions in PAO1 Δ5\u0026rsquo;UTR (see Supplementary Figure S3a), while NrdJ protein levels appear increased under both aerobic and anaerobic conditions (see Supplementary Figure S3c). Additionally, \u003cem\u003enrdJ\u003c/em\u003e expression seems to be reduced during \u003cem\u003eG. mellonella\u003c/em\u003e infection (see Supplementary Figure S3b).\u003c/p\u003e\u003cp\u003eTherefore, this imbalance produced by the absence of the 5\u0026rsquo; UTR may impair the coordinated regulation of the ribonucleotide reductase (RNR) system between \u003cem\u003enrdA\u003c/em\u003e and \u003cem\u003enrdJ\u003c/em\u003e, rendering the ∆5\u0026rsquo;UTR strain less fit and, consequently, less virulent (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStrains, plasmids, and growth conditions\u003c/h2\u003e\u003cp\u003eThe bacterial strains and the plasmids used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e strains were routinely grown in Luria-Bertani (Scharlab, Spain) medium or minimal medium (MM)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e at 37\u0026ordm;C. For anaerobic growth, LB medium containing KNO\u003csub\u003e3\u003c/sub\u003e (10 g/L) (LBN) was used in screw-cap Hungate tubes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Liquid cultures were shaken at 200 rpm. Antibiotics were added at the following concentrations: 50 \u0026micro;g/mL ampicillin and 10 \u0026micro;g/mL gentamicin for \u003cem\u003eE. coli\u003c/em\u003e; and 100 \u0026micro;g/mL gentamicin, 40 \u0026micro;g/mL tetracycline, and 300 \u0026micro;g/mL carbenicillin for \u003cem\u003eP. aeruginosa\u003c/em\u003e. For transcriptional and translational assays, rifampicin (Sigma Aldrich, Merck) was used at a final concentration of 200 \u0026micro;g/mL, along with variable concentrations of adenosyl cobalamin or coenzyme B\u003csub\u003e12\u003c/sub\u003e (Sigma Aldrich, Merck), depending on the experiment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDNA manipulation\u003c/h3\u003e\n\u003cp\u003eDNA manipulation and plasmid constructions were performed using standard protocols \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. All kits and molecular biology enzymes used were obtained from Thermo Fisher Scientific (Spain) and were used according to the manufacturer\u0026rsquo;s instructions. DNA fragments were amplified using Phusion High-Fidelity DNA polymerase or DreamTaq Green PCR MasterMix with the primers listed in Supplementary Table S2. DNA fragments were isolated and purified from agarose gels using a GeneJet Gel Extraction kit (Thermo Fisher Scientific) and Monarch\u0026reg; DNA gel extraction kit (New England Biolabs) in the case of digested DNA. Plasmid DNA was extracted using a GeneJET Plasmid Miniprep kit (Thermo Fisher Scientific) and transferred into \u003cem\u003eP. aeruginosa\u003c/em\u003e cells via electroporation with a Gene Pulser XCell electroporator (Bio-Rad) as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. All the constructs obtained were verified by DNA sequencing by Eurofins Genomics.\u003c/p\u003e\n\u003ch3\u003ePlasmid construction\u003c/h3\u003e\n\u003cp\u003eThe plasmids pETS257, pETS258, and pETS259 were constructed as follows (See Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Briefly, the \u003cem\u003enrdA\u003c/em\u003e promoter region was amplified using the primers 1 and 5, for the P\u003cem\u003enrdA\u003c/em\u003e translational fusion (pETS258); 1 and 3, and 2 and 4 for transcriptional P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR \u003cem\u003egfp\u003c/em\u003e fusion (pETS257); 1 and 3, and 4 and 5 for P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR \u003cem\u003egfp\u003c/em\u003e translational fusion (pETS259). For P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR constructs, two fragments were generated from primers described before and used as templates for overlap extension PCR with primers 1 and 2. The GFP gene (green fluorescent protein) was amplified from pETS130 using 7 and 8 primers. The \u003cem\u003egfp\u003c/em\u003e insert, P\u003cem\u003enrdA\u003c/em\u003e and P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR inserts were digested with the \u003cem\u003eSma\u003c/em\u003eI restriction enzyme and ligated with T4 ligase to generate P\u003cem\u003enrdA\u003c/em\u003e and P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR GFP translational fusions. The resulting fragments (P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR, P\u003cem\u003enrdA\u003c/em\u003e-Δ5\u0026rsquo;UTR::\u003cem\u003egfp\u003c/em\u003e, P\u003cem\u003enrdA\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e, were cloned separately to pJET 1.2 vector (Thermo Scientific) and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5a. The resulting plasmids and pETS130-GFP vector were digested with \u003cem\u003eBam\u003c/em\u003eHI-\u003cem\u003eKpn\u003c/em\u003eI (translational GFP fusions) or \u003cem\u003eBam\u003c/em\u003eHI-\u003cem\u003eSma\u003c/em\u003eI (transcriptional GFP fusion) and ligated using the T4 ligase enzyme. These plasmids were electroporated into \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and transcriptional GFP fusion plasmids were also electroporated into \u003cem\u003eP. aeruginosa\u003c/em\u003e Δ\u003cem\u003erne\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e Δ\u003cem\u003erhl\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e Δ\u003cem\u003ernr.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe plasmids pJN106 and pJN107 were generated as described below (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 5\u0026rsquo;UTR forward region was amplified by primers 9 and 10, while the 5\u0026rsquo;UTR reverse orientation was amplified by primers 11 and 12. The resulting fragments, named 5\u0026rsquo;UTRfor and 5\u0026rsquo;UTRrev, were cloned separately to pJET 1.2 and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5a. The resulting plasmids and pJN105 vector were digested with \u003cem\u003eEco\u003c/em\u003eRI-\u003cem\u003ePst\u003c/em\u003eI and ligated using T4 ligase enzyme. These plasmids were electroporated into \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1.\u003c/p\u003e\n\u003ch3\u003eCRISPR-cas9 mutant construction\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR (\u003cem\u003enrdA\u003c/em\u003e) chromosomal deletion mutant was generated using the CRISPR-cas9 toolkit described before\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, following the established protocol with some modifications. The recombineering ssDNA oligonucleotide consisted of 50 nucleotides downstream of the transcriptional starting site (located 409 bp upstream of the \u003cem\u003enrdA\u003c/em\u003e start codon-ATG), directly followed by 50 nucleotides downstream of the \u003cem\u003enrdA\u003c/em\u003e ribosome binding site (located 9 bp upstream of the start codon). The oligonucleotide was synthesized by Eurofins Genomics (see Supplementary Table S2).\u003c/p\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 pSH124 cells were electroporated with both plasmids pS149 (pS148-Δ5\u0026rsquo;UTR) and the recombineering oligonucleotide (5\u0026rsquo;UTR_PAO1(+)-Rec) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). Screening of edited strains was performed using two consecutive colony PCR (cPCR): an initial cPCR was conducted on pools of colonies, and upon identification of a positive pool, individual colonies were subsequently tested by cPCR. Positive colonies were sequenced by Eurofins Genomics, and the plasmids were subsequently cured as described in the protocol.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGene reporter assay experiments\u003c/h2\u003e\u003cp\u003eGreen fluorescent protein reporter assay was carried out with two methodologies; first, \u003cem\u003eP. aeruginosa\u003c/em\u003e bacterial pETS130-GFP derivatives growth was monitored by measuring optical density at 600 nm wavelength (OD\u003csub\u003e600\u003c/sub\u003e) on growth medium (LB for aerobic cultures and LBN for anaerobic cultures) at 37\u0026deg;C and 200 rpm to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.4\u0026ndash;0.7 (Exponential) and OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;2 (Stationary) where three independent 1 ml samples were taken. Samples were centrifuged at 6000 rpm for 10 min. The pellet was washed with phosphate-buffered saline (PBS) (Fisher Scientific) solution containing 2% formaldehyde and stored in the dark at 4\u0026deg;C for 10 min. The samples were centrifuged again, and the pellets were resuspended in 1 mL PBS. The fluorescence of the samples was measured in 96-well plates (Costar \u0026reg; 96-Well Black polystyrene plate, Corning) in an Infinite 200 Pro Fluorescence Microplate Reader (Tecan, Switzerland).\u003c/p\u003e\u003cp\u003eAlternatively, \u003cem\u003eP. aeruginosa\u003c/em\u003e bacterial pETS130-GFP derivatives growth and GFP fluorescence was monitored simultaneously every 20\u0026ndash;30 minutes in a 96-well plate (Costar \u0026reg; 96-Well Black polystyrene plate, Corning) in a Spark microplate reader (Tecan, Switzerland) at 37\u0026ordm;C.\u003c/p\u003e\u003cp\u003eFor the riboswitch test, \u003cem\u003eP. aeruginosa\u003c/em\u003e strains containing derivatives of the pETS130-GFP plasmid were grown in minimal medium (MM)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.3. Different concentrations of B\u003csub\u003e12\u003c/sub\u003e vitamin were added (1 mg/ml, 2.5 mg/mL and 10 mg/mL) and GFP expression was measured in a 96-well plate (Costar \u0026reg; 96-Well Black polystyrene plate, Corning) every 20 min in an Infinite 200 Pro Fluorescence Microplate Reader (Tecan, Switzerland).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern immunoblot analysis\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 WT and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 Δ5\u0026rsquo;UTR strain cultures were collected under aerobic and anaerobic conditions during exponential (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.4\u0026ndash;0.7) and stationary (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;2) growth phases. Protein concentrations were adjusted to 10 \u0026micro;g for all the samples based on quantification using the Bradford Assay (Bio-Rad), with bovine serum albumin (BSA) as the standard. Western blotting was conducted as previously described, with some modifications\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. A 1:10000 dilution of polyclonal Anti-NrdA or Anti-NrdJ primary antibodies (Agrisera, Sweden; and Thermo Fisher, USA) was used, followed by detection with a donkey anti-rabbit horseradish peroxidase\u0026ndash;conjugated secondary antibody (Bio-Rad) at a 1:750 dilution. The antibody\u0026ndash;antigen complexes were visualized using Amersham\u0026trade; ECL\u0026trade; Prime Western Blotting Reagent (GE Healthcare), according to the manufacturer\u0026rsquo;s instructions. Protein bands were visualized and analysed by the ImageQuant\u0026trade; LAS 4000 Mini system (GE Healthcare). The resulting image was processed using Fiji (ImageJ, NIH, USA) for visualization purposes.\u003c/p\u003e\n\u003ch3\u003e5’UTR overexpression\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 pJN106 and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 pJN107 cultures were collected during the exponential phase and after 2 hours of induction with 0.4% L-arabinose. Pellets were lysed with BugBuster\u0026reg; 1X (Sigma Aldrich) according to the manufacturer\u0026rsquo;s instructions. The lysates were centrifuged at 16000g for 20 minutes at 4 \u0026ordm;C. Protein concentrations were determined by the Bradford assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. A 4\u0026ndash;20% Mini-PROTEAN\u0026reg; TGX\u003csup\u003e\u0026trade;\u003c/sup\u003e Precast protein gel (Bio-Rad) was loaded, and the gel was run at 40 mA.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNA isolation, reverse transcription and real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 strain cultures were collected under aerobic and anaerobic conditions during exponential (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.4\u0026ndash;0.7) and stationary (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;2) growth phases. Total RNA was extracted using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. To further remove DNA contamination, the RNA was treated with TURBO\u0026trade; DNase (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions and verified by PCR with the primers listed in Supplementary Table S2. The amount of RNA was determined using a NanoDrop (Nanodrop spectrophotometer ND-1000). The cDNA retro-transcription was performed using Maxima Reverse Transcriptase (Thermo Fisher Scientific) and Random hexamers (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR measurements were conducted using PowerUp\u0026trade; SYBR\u003csup\u003e\u0026minus;\u003c/sup\u003eGreen\u0026trade; (Applied Biosystems) with the SYBR-Green listed in Supplementary Table S2, and detection was performed using a StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems) following the manufacturer\u0026rsquo;s specifications. The \u003cem\u003egap\u003c/em\u003e gene was used as an internal standard unless otherwise specified. The results were analysed using the ΔΔCt method unless stated differently.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePrimer extension\u003c/h2\u003e\u003cp\u003eBacterial cells were grown in LB medium to an OD\u003csub\u003e600\u003c/sub\u003e of 0.7, and the total RNA was extracted with GeneJET RNA extraction kit (Thermo Fisher Scientific) following the same protocol as Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR). SupersScript reverse transcriptase (Thermo Fisher Scientific) was used to reverse transcribe \u003cem\u003enrdA\u003c/em\u003e using extension primers 25 and 26 (See Supplementary Table S2). The primer was radioactively labelled with [g-\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP using T4 Polynucleotide Kinase (Thermo Fisher Scientific). A DNA fragment of 841 nucleotides corresponding to the sequence upstream of the \u003cem\u003enrdA\u003c/em\u003e coding region was amplified by PCR using High Expand Taq polymerase (Roche). The resulting PCR products generated a sequence ladder with the same primer used for the extension reaction using the Sequenase kit (Promega). The extension product was resolved on an 8% acrylamide \u0026ndash; 7M urea gel alongside the sequence ladders.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5\u0026rsquo;RACE (5\u0026rsquo; Rapid alignment of cDNA ends)\u003c/h2\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 cultures were collected under aerobic and anaerobic conditions during the exponential growth phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5), and RNA extraction was performed as previously described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR). cDNA synthesis was performed using Maxima Reverse Transcriptase (Thermo Fisher Scientific), according to the manufacturer\u0026rsquo;s instructions. Each reaction contained 1.5 pmol of gene-specific primer (44, Supplementary Table S2) and 0.3 mg of total RNA. The resulting cDNA was purified using the High Pure PCR Product Purification Kit (Roche Life Science), according to the manufacturer\u0026rsquo;s instructions. Poly-A tailing was performed using 40 U of Terminal Deoxynucleotidyl Transferase (Thermo Fisher Scientific) and 1.5 nmol dATP (Thermo Fisher Scientific).\u003c/p\u003e\u003cp\u003eThe first PCR (PCR1) was conducted using primers45 and 24 (Supplementary Table S2) with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific), using the poly-A cDNA as a template. PCR1 was optimised with 10 cycles of 40-second extension, followed by 25 cycles with a 20-second extension, increasing by 5 seconds per cycle. The PCR1 product was gel-purified using the GeneJet Gel Extraction kit (Thermo Fisher Scientific) and diluted 1:20. The second PCR (PCR2) was performed using primers 46 and 47 (Supplementary Table S2), with PCR1 as a template, using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. PCR2 products were purified and cloned into the pJET1.2 blunt vector, following the same protocol as described in Materials and Methods Section Plasmid construction). Colonies were sequenced by Eurofins Genomics using the primer 47 (Supplementary Table S2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptional shut-off assay\u003c/h2\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 \u003cem\u003ewild-type\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e Δ5\u0026rsquo;UTR \u003cem\u003eP. aeruginosa\u003c/em\u003e pETS134, and \u003cem\u003eP. aeruginosa\u003c/em\u003e pETS257 strains were grown and treated with 200 \u0026micro;g/ml of rifampicin (a transcriptional blocking agent). Samples were collected at 0, 5, and 20 minutes after rifampicin addition to arrest transcription for \u003cem\u003eP. aeruginosa\u003c/em\u003e pETS134 and pETS257, and at 0, 2, 6, 10, and 20 minutes for \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 \u003cem\u003ewild-type\u003c/em\u003e, and Δ5\u0026rsquo;UTR. Total RNA was extracted using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Reverse transcription and subsequent quantitative real-time PCR (qRT-PCR) measurements were performed as described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR), with the modification that the 16S rRNA gene was used as an internal control and \u003cem\u003enrdJ\u003c/em\u003e as the reference gene.\u003c/p\u003e\u003cp\u003eAbsolute PCR quantification was performed using a standard curve from target genes (\u003cem\u003ecat\u003c/em\u003e, \u003cem\u003enrdA\u003c/em\u003e, \u003cem\u003enrdJ\u003c/em\u003e, 16S). Briefly, PCR amplification of genomic \u003cem\u003enrdA\u003c/em\u003e, \u003cem\u003enrdJ\u003c/em\u003e, and 16S rRNA gene, and \u003cem\u003ecat\u003c/em\u003e from pETS130-GFP plasmid was performed using the primers listed on Supplementary Table S2. A ten-fold dilution of known amplicons quantities was performed, and the number of copies was calculated by the following formula (Integrated DNA Technologies, IDT):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Number\\:of\\:copies=\\frac{Quantity\\:of\\:DNA\\:\\left(ng\\right)\\times\\:6.022\\times\\:{10}^{23}}{Length\\:of\\:DNA\\:\\left(bp\\right)\\times\\:1\\times\\:{10}^{9}\\times\\:650}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn which \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:6.022\\times\\:{10}^{23}\\)\u003c/span\u003e\u003c/span\u003eis Avogadro number, 650 is the average weight (grams, g) of a base pair (bp) in Daltons and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1\\times\\:{10}^{9}\\)\u003c/span\u003e\u003c/span\u003e is to convert grams(g) to nanograms (ng).\u003c/p\u003e\u003cp\u003eOnce the Ct values from each dilution of every target gene were determined, a standard curve was established by plotting the target DNA copy number against each Ct value. The standard curve was used to calculate the number of copies from each target gene in different samples. The percentage of mRNA retro-transcribed remaining was calculated using non-rifampicin treated sample or the before-rifampicin sample as the initial mRNA amount of each target gene.\u003c/p\u003e\u003cp\u003eData were analyzed using nonlinear regression analysis in GraphPad Prism 10.1.1 (GraphPad Software). A one-phase decay model was applied assuming a decay from a shared initial value (Y\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;100). The equation model was: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Y=\\left({Y}_{0}-Plateau\\right)\\times\\:{e}^{-K\\times\\:X}+Plateau\\)\u003c/span\u003e\u003c/span\u003e, where X is time (minutes) and Y is percentage (%) of remaining cDNA that decays with one phase down to a Plateau, and K is the decay rate constant.\u003c/p\u003e\u003cp\u003eTo ensure biological plausibility and stability of the fit, constraints were applied: K\u0026thinsp;\u0026gt;\u0026thinsp;0.01 and in some cases, Plateau values were fixed when instability was detected in the fitting process. Key parameters extracted from the fit included the decay rate constant (K) and half-life (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{t}_{1/2}=\\frac{(\\text{ln}2)}{K}\\)\u003c/span\u003e\u003c/span\u003e). For the sample \u003cem\u003enrdA\u003c/em\u003e transcript sample within WT\u0026thinsp;+\u0026thinsp;P\u003cem\u003enrdA\u003c/em\u003e- Δ5\u0026rsquo;UTR parameters determination also included fitting the plateau (Plateau\u0026thinsp;=\u0026thinsp;6) to ensure that the calculated values accurately reflect the true behaviour of the system. Comparisons between the groups focused on differences in K and half-life as indicators of decay rate.\u003c/p\u003e\u003cp\u003eThe goodness of the fit was evaluated using the R\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e value and 95% confidence intervals (profile likelihood method). In some cases (Δ5\u0026rsquo;UTR, \u003cem\u003enrdJ\u003c/em\u003e transcript), profile likelihood intervals could not be fully determined and were calculated using the symmetrical (asymptotic) method implemented in GraphPad Prism 10.1.1 (GraphPad Software). While this method assumes normality and may underestimate the uncertainty for some parameters, it provides a consistent basis for comparison.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGalleria mellonella\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eGalleria mellonella\u003c/em\u003e was maintained and dose-infected as described before\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e with some modifications. The different doses were plated in LB (Scharlab) plates to determine bacterial CFU (20\u0026ndash;40 CFUs per larva).\u003c/p\u003e\u003cp\u003eRegarding bacterial RNA extraction during infection, groups of 10 bacterial-infected larvae were collected about 16-17h after the course of infection and anesthetized on ice for 10 min. Bacterial sample collection from \u003cem\u003eGalleria mellonella\u003c/em\u003e was performed following the protocol described before \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and RNA extraction was performed as described in Materials and Methods Section RNA isolation, reverse transcription and real-time PCR (qRT-PCR).\u003c/p\u003e\u003cp\u003eIn the case of \u003cem\u003eGalleria mellonella\u003c/em\u003e virulence analysis, groups of 10 larvae were infected and kept at 37\u0026ordm;C and periodically monitored during infection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatic analysis and data sourcing\u003c/h2\u003e\u003cp\u003eRiboswitch prediction analysis was performed using Riboswitch Scanner\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e with default settings. RegPrecise\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e was used to identify cobalamin regulon genes in \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. Sequences obtained from RegPrecise database and putative B\u003csub\u003e12\u003c/sub\u003e riboswitch sequences were aligned using T-coffee software\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e with default parameters. The alignment was manually analysed, considering the predicted secondary structure.\u003c/p\u003e\u003cp\u003eBLAST software \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and the Coding Potential Calculator 2 (CPC2) tool\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e were used for protein-coding potential prediction, both with default settings. The Rfam\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and RNAcentral\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e databases were used to submit 5\u0026rsquo;UTR sequence to identify potential matches with non-coding RNA sequences. The presence of Rho-independent terminators was evaluated using ARNold software\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e with standard settings.\u003c/p\u003e\u003cp\u003ePromoter prediction was conducted using PromoterHunter\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and BPROM\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Both scientific publications and data repositories were consulted, including Kyoto Encyclopaedia of genes and genomes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, as well as raw data from several RNA-seq experiments, with the specific sources cited in the relevant sections of the manuscript.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 10.1.1 (GraphPad Software). Single comparisons were performed using unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Statistical significance was indicated by \u003cem\u003ep\u003c/em\u003e-values as follows: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Error bars in figures represent the standard deviation (SD) between samples. Welch's t-test was applied when unequal variances were detected.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe 5\u0026rsquo; UTR appears to regulate \u003cem\u003enrdAB\u003c/em\u003e mRNA transcription and stability and may serve as a molecular mechanism to down-regulate \u003cem\u003enrdAB\u003c/em\u003e expression during the stationary phase, when the synthesis of dNTPs is no longer required for DNA replication. To date, no mechanisms have been described in \u003cem\u003eP. aeruginosa\u003c/em\u003e that explain how \u003cem\u003enrdA\u003c/em\u003e transcription is down-regulated upon entry into stationary phase.\u003c/p\u003e\u003cp\u003eThe absence of the 5\u0026rsquo; UTR from the \u003cem\u003enrdAB\u003c/em\u003e class Ia RNR operon in \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 led to increased transcription, enhanced mRNA stability and greater structural flexibility (Figure. 8). These changes ultimately result in higher NrdA protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This effect is likely due to the increased number of \u003cem\u003enrdAB\u003c/em\u003e mRNA copies and their improved stability, which together enhance ribosome accessibility and translation efficiency, rather than an increase in translation initiation itself.\u003c/p\u003e\u003cp\u003eHowever, instead of conferring an adaptive advantage, the loss of the 5\u0026rsquo; UTR appears to disrupt the normal regulatory balance between RNR classes (\u003cem\u003enrdA\u003c/em\u003e and \u003cem\u003enrdJ\u003c/em\u003e), leading to reduced virulence of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 during \u003cem\u003eGalleria mellonella\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings highlight the crucial role of the 5\u0026rsquo;UTR in fine-tuning \u003cem\u003enrdAB\u003c/em\u003e operon expression and underscore the importance of untranslated regions as regulatory elements. Nevertheless, further studies are needed to elucidate the molecular mechanisms underlying \u003cem\u003enrdAB\u003c/em\u003e modulation and its integration into broader regulatory networks.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank to Dr. Susanne H\u0026auml;u\u0026szlig;ler and Dr. Alejandro Arce-Rodriguez for kindly providing CRISPR-Cas9 toolkit plasmids and for their technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially supported by grants\u0026nbsp;PID2021-125801OB-100,\u0026nbsp;PLEC2022-009356 and PDC2022-133577-I00, funded by MCIN/AEI/ 10.13039/501100011033 and \u0026ldquo;ERDF A way of making Europe\u0026rdquo;, the CERCA programme, and \u003cem\u003eAGAUR-Generalitat de Catalunya\u003c/em\u003e (2021SGR01545), the European Regional Development Fund (FEDER), the Catalan Cystic Fibrosis association, and Obra Social \u0026ldquo;La Caixa\u0026rdquo;. E.T. is a researcher of the ICREA Academia 2025 program. A.M-M thanks Generalitat de Catalunya for its financial support through the FI program (FI_B00313).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.M-M and ET wrote the manuscript. A.M-M, AR and LP performed the biological assays. ET supervised research, managed the project, secured funding, and reviewed the experimental data. All authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study, including full-length original gels and blots, are available in the Supplementary Material and at the public repository: https://doi.org/10.34810/data2361.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLetizia, M., Diggle, S. 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Identification of small non-coding RNAs from \u003cem\u003eRhizobium\u003c/em\u003e \u003cem\u003eetli\u003c/em\u003e by integrated genome wide and transcriptome-based methods. \u003cem\u003eExRNA\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1\u0026ndash;11 (2020).\u003c/li\u003e\n \u003cli\u003eG\u0026oacute;mez-Lozano, M. \u003cem\u003eet al.\u003c/em\u003e Diversity of small RNAs expressed in \u003cem\u003ePseudomonas\u003c/em\u003e species. \u003cem\u003eEnviron Microbiol Rep\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 227\u0026ndash;236 (2015).\u003c/li\u003e\n \u003cli\u003eXia, Y. \u003cem\u003eet al.\u003c/em\u003e Endoribonuclease Ybey is essential for RNA processing and virulence in \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;21 (2020).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ribonucleotide reductase, 5’UTR, mRNA stability, transcription, protein levels, Galleria mellonella","lastPublishedDoi":"10.21203/rs.3.rs-6937143/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6937143/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe class Ia ribonucleotide reductase (\u003cem\u003enrdAB\u003c/em\u003e) operon in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e encodes a long 5\u0026rsquo; untranslated region (5\u0026rsquo;UTR), whose regulatory role remains poorly understood. In this study, we studied the functional significance of the \u003cem\u003enrdAB\u003c/em\u003e 5\u0026rsquo;UTR through a combination of comprehensive set of bioinformatic and experimental approaches combining gene expression studies, protein analysis, and infection in \u003cem\u003eGalleria mellonella in vivo\u003c/em\u003e animal model. Our results demonstrate that the 5\u0026rsquo;UTR negatively regulates \u003cem\u003enrdA\u003c/em\u003e expression by reducing transcription and decreasing mRNA stability. Deletion of the 5\u0026rsquo;UTR led to increased \u003cem\u003enrdA\u003c/em\u003e 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\u0026rsquo;UTR may lead to a more flexible mRNA conformation, which could facilitate ribosome accessibility. Notably, this deregulation disrupted the balance between \u003cem\u003enrdA\u003c/em\u003e and \u003cem\u003enrdJ\u003c/em\u003e expression, leading to reduced virulence in a \u003cem\u003eG. mellonella\u003c/em\u003e infection model. This effect results from a slight decrease in \u003cem\u003enrdJ\u003c/em\u003e expression combined with an increase in \u003cem\u003enrdA\u003c/em\u003e mRNA levels, which may compromise the optimal RNR regulation during infection. These findings highlight the 5\u0026rsquo;UTR as a key regulatory element in fine-tuning \u003cem\u003enrdAB\u003c/em\u003e expression and maintaining RNR system homeostasis in \u003cem\u003eP. aeruginosa.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"The long 5′UTR of nrdAB modulates transcription, mRNA stability, and virulence in Pseudomonas aeruginosa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 14:31:04","doi":"10.21203/rs.3.rs-6937143/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-18T03:32:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T11:06:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-12T10:25:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163771795539729621438079147238965690040","date":"2025-07-08T12:58:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79843052084894451075096804056289935561","date":"2025-07-08T11:41:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-08T09:49:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-08T09:36:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-08T04:00:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-29T14:46:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-29T14:43:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3264b45c-209e-4eb2-b900-5c956c1bdb4e","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51290233,"name":"Biological sciences/Microbiology/Bacteria/Bacterial genetics"},{"id":51290235,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"},{"id":51290238,"name":"Biological sciences/Microbiology/Bacteria/Bacterial transcription"}],"tags":[],"updatedAt":"2025-10-20T16:00:20+00:00","versionOfRecord":{"articleIdentity":"rs-6937143","link":"https://doi.org/10.1038/s41598-025-20079-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-15 15:57:15","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-07-11 14:31:04","video":"","vorDoi":"10.1038/s41598-025-20079-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-20079-6","workflowStages":[]},"version":"v1","identity":"rs-6937143","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6937143","identity":"rs-6937143","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}