Abstract
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.
Highlights
In Gram negative bacteria, at least 30% of proteins are localized outside the cytoplasm, where they are produced[1]
The P. aeruginosa proteome has been manually screened for the presence of Tat signal peptides in two studies either looking for the motif RRXFLK/R33 or the two conserved arginines followed by at least two of the FLK/R residues[37,38]
To avoid dismissing real Tat substrates predicted only by TATFIND or TatP as occurred in Streptomyces coelicolor[22], we compared our list of candidates with the candidates obtained with the most recent program, PRED-TAT
Summary
In Gram negative bacteria, at least 30% of proteins are localized outside the cytoplasm, where they are produced[1]. Signal peptides have a tripartite organization with a positively charged N-terminal region (N-region), a central hydrophobic core (H-region) and a polar C-terminal region (C-region)[6] Despite their similar overall organization, Sec and Tat signal peptides have characteristics that allow specific recognition by their respective export pathway. The C-region of Tat signal peptides contains conserved motifs which are recognition sequences for signal peptidases I and II8,9 as well as basic residues that impede targeting to the Sec pathway[10]. PRED-TAT uses a Hidden Markov Model (HMM) method with sub-modules for the distinguishing between Sec, Tat and TM (transmembrane) regions Comparisons of these prediction algorithms on test sets tend to show that TatP generates fewer false positives than TATFIND and slightly more false negatives[13]. This is mainly because the full repertoire of Tat proteins in P. aeruginosa is unknown
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