Novel Twin-Arginine Translocation Pathway-Dependent Phenotypes of Bacillus subtilis Unveiled by Quantitative Proteomics

Abstract

The twin-arginine translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal, and organellar membranes. To date, the mechanisms involved in processing, proofreading, and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane, and extracellular fractions were analyzed by LC-MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein-protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

“…As previously shown by subcellular fractionation, the intact QcrA is an 18-kDa membrane-associated protein with N in -C out topology (28). Importantly, the translocated QcrA is exposed to signal peptidase activity, resulting in the release of a 14-kDa processed form (QcrA*) into the growth medium (Fig.…”

“…QcrA-C123S). Membrane insertion and translocation of these mutant QcrA forms was assessed by Western blotting, taking advantage of the fact that translocated QcrA is to some extent processed by signal peptidase, which results in the release of a smaller form (QcrA*) into the growth medium (28). It is presently not known why QcrA is processed by signal peptidase and why QcrA* is released into the medium, especially because the known QcrA biological function is electron transfer via the cytochrome bc 1 complex the cytoplasmic membrane.…”

“…Subcellular Fractionations-Subcellular fractionation studies were performed as described previously (28). However, for the present experiments cultures were grown to early stationary phase.…”

“…Polyclonal antibodies specific for BdbD, LipA, QcrA, and TrxA have been described previously (28,59,60). Bound antibodies were detected with fluorescent IgG secondary antibodies (IRDye 800 CW goat anti-rabbit from LiCor Biosciences) and visualized at 700 or 800 nm with the Odyssey Infrared Imaging System (LiCor Biosciences).…”

“…1a). In fact, the processing of QcrA was only abolished when four of the five type I signal peptidase-encoding genes of B. subtilis were deleted (28). Because the catalytic site of signal peptidase is positioned on the extracytoplasmic side of the membrane, QcrA processing and release of QcrA* into the medium can be used as a read-out for the translocation of this protein.…”