Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane.

Institute of Biochemistry and Molecular Biology, Zentrum für Biochemie und Molekulare Zellforschung, University of Freiburg, 79104 Freiburg, Germany.
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2011; 287(4):2591-9. DOI: 10.1074/jbc.M111.293068
Source: PubMed

ABSTRACT The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of chaperone-usher pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a non-chaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (polypeptide transport associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are co-regulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain. This article is protected by copyright. All rights reserved.
    Protein Science 01/2015; 24(5). DOI:10.1002/pro.2640 · 2.86 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Outer membrane proteins are vital for Gram-negative bacteria and organisms which inheri-ted organelles from them. Proteins from the Omp85/BamA family carry out the insertion of membrane proteins into the outer membrane. We show that an 8-stranded outer mem¬brane β-barrel protein, TtoA, is inserted and folded into liposomes by an Omp85 homo¬logue. Furthermore, we recorded the channel conduc¬tance of this Omp85 protein in black-lipid mem¬branes, alone and in presence of peptides comprising the sequence of the two N- and the two C-terminal β-strands of TtoA. Only with the latter a long-living com¬pound channel could be observed that exhibits larger conduc¬tance levels than the Omp85 protein alone. These data support a model where unfolded outer membrane pro¬tein after docking with its C-terminus, penetrates into the transmembrane β-barrel of the Omp85 protein and aug-ments its β-sheet at the first strand. Augmentation with successive β-strands leads to a com-pound, dilated barrel of both proteins.
    Biochemistry 12/2014; 54(3). DOI:10.1021/bi5011305 · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gram-negative bacteria use the type-V secretion pathway to expose proteins at their cell surface, many of which have virulence functions. Translocation of those proteins across the outer membrane occurs either by means of dedicated translocator proteins (two-partner secretion) or covalently fused translocator domains (autotransporters). Translocator proteins and translocator domains are β-barrels requiring the β-barrel assembly machinery (BAM) for membrane integration. However, the molecular details of their passage across the envelope and insertion into the outer membrane remain enigmatic, owing in part to the fact that in vitro systems are not available. Here we describe a versatile in vitro reconstitution system that faithfully reproduces both branches of the type-V secretion pathway and the assembly of β-barrel outer membrane proteins. This system will allow an in-depth analysis of protein secretion across and integration into outer membranes.
    Nature Communications 11/2014; 5:5396. DOI:10.1038/ncomms6396 · 10.74 Impact Factor