2 research outputs found
Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa
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
Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa
Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 degrees C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa. (C) 2009 Elsevier B.V. All rights reserved