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In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we performed a combination of in silico analysis and experimental validation to identify a core set of Tat substrates in the opportunistic pathogen Pseudomonas aeruginosa. In silico analysis predicted 44 putative Tat signal peptides in the P. aeruginosa PA14 proteome. We developed an improved amidase-based Tat reporter assay to show that 33 of these are real Tat signal peptides. In addition, in silico analysis of the full translated genome revealed a Tat candidate with a missassigned start codon. We showed that it is a new periplasmic protein in P. aeruginosa. Altogether we discovered and validated 34 Tat substrates. These show little overlap with Escherichia coli Tat substrates, and functional analysis points to a general role for the P. aeruginosa Tat system in the colonization of environmental niches and pathogenicity.

The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell. However, the full range of proteins that are conveyed by Tat is unknown, despite the importance of Tat for the adaptability and full virulence of P. aeruginosa. In this work, we explored the P. aeruginosa Tat-dependent exoproteome under phosphate starvation by two-dimensional gel analysis. We identified the major secreted proteins and new Tat-dependent exoproteins. These exoproteins were further analyzed by a combination of in silico analysis, regulation studies, and protein localization. Altogether we reveal that the absence of the Tat system significantly affects the composition of the exoproteome by impairing protein export and affecting gene expression. Notably we discovered three new Tat exoproteins and one novel type II secretion substrate. Our data also allowed the identification of two new start codons highlighting the importance of protein annotation for subcellular predictions. The new exoproteins that we identify may play a significant role in P. aeruginosa pathogenesis, host interaction and niche adaptation.