Article

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.

0 Followers
 · 
133 Views
  • [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
  • [Show abstract] [Hide abstract]
    ABSTRACT: TpsB proteins are Omp85 superfamily members that mediate protein translocation across the outer membrane of Gram-negative bacteria. Omp85 transporters are composed of N-terminal POTRA domains and a C-terminal transmembrane β-barrel. In this work, we track the in vivo secretion path of the Bordetella pertussis filamentous haemagglutinin (FHA), the substrate of the model TpsB transporter FhaC, using site-specific crosslinking. The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner β-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded β-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding. This work demonstrates that the Omp85 barrel serves as a channel for translocation of substrate proteins.
    Nature Communications 10/2014; 5:5271. DOI:10.1038/ncomms6271 · 10.74 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