Comparative genomics has greatly facilitated the identification of shared as well as unique features among individual cells or tissues, and thus offers the potential to find disease markers. While proteomics is recognized for its potential to generate quantitative maps of protein expression, comparative proteomics in bacteria has been largely restricted to the comparison of single cell lines or mutant strains. In this study, we used a data independent acquisition (DIA) technique, which enables global protein quantification of large sample cohorts, to record the proteome profiles of overall 27 whole genome sequenced and transcriptionally profiled clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. Analysis of the proteome profiles across the 27 clinical isolates grown under planktonic and biofilm growth conditions led to the identification of a core biofilm-associated protein profile. Furthermore, we found that protein-to-mRNA ratios between different P. aeruginosa strains are well correlated, indicating conserved patterns of post-transcriptional regulation. Uncovering core regulatory pathways, which drive biofilm formation and associated antibiotic tolerance in bacterial pathogens, promise to give clues to interactions between bacterial species and their environment and could provide useful targets for new clinical interventions to combat biofilm-associated infections.

Summary: Systems biology approaches address the challenge of translating sequence information into function. In this study, we described the Pseudomonas aeruginosa PA14 proteomic landscape and quantified environment‐driven changes in protein levels by the use of LC–MS techniques. Previously recorded mRNA data allowed a comparison of RNA to protein ratios for each individual gene and, thus, to explore the relationship between an mRNA being differentially expressed between environmental conditions and the mRNA‐protein correlation for that gene. We developed a Random Forest‐based predictor for protein levels and found that the mRNA to protein correlation was higher for genes/proteins that undergo dynamic changes. One example of a discrepancy between protein and predicted protein levels was observed for a phage‐related gene cluster, which was translated into low protein levels under standard growth conditions. However, under SOS‐inducing conditions more protein was produced and the prediction of protein levels based on mRNA abundancy became more accurate. In conclusion, our systems biology approach sheds light on complex mRNA to protein level relationships and uncovered condition‐dependent post‐transcriptional regulatory events.

Post-transcriptional modification of transfer RNAs (tRNAs) represents an essential layer of translational regulation critical for bacterial adaptation to environmental changes. Increasing evidence links the tRNA epitranscriptome to pivotal roles in the regulation of gene expression and various cellular processes, including stress responses and establishment of virulence. In this study, we used mass spectrometry and nanopore sequencing to quantify and identify the sites of TrhPO-dependent tRNA hydroxylation in total and purified Pseudomonas aeruginosa tRNAs. Furthermore, transcriptome, ribosome profiling, and proteome data were integrated to demonstrate the post-transcriptional consequences of the absence of xo5U34 modifications at the wobble position of selected tRNAs. We suggest that the impaired ability to infect host cells and attenuated virulence in Galleria mellonella are driven by changes in metabolic fluxes. In the absence of TrhPO-mediated tRNA modification, chorismate, the precursor for the biosynthesis of xo5U modifications, is funneled into alternative pathways, including the production of aromatic amino acids and phenazines. Our findings that metabolic rerouting, rather than changes in proteome profiles, attenuates P. aeruginosa virulence highlight the multifunctional roles of tRNA-modifying enzymes and suggest an underexplored role for these enzymes in monitoring and modulating metabolic fitness. These insights open new avenues for combatting the pathogenicity of this challenging opportunistic pathogen.