Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane.
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.
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ABSTRACT: FhaC is an outer membrane transporter from Bordetella pertussis belonging to the two-partner secretion (TPS) pathway with its primary role being the secretion of the virulence factor filamentous haemagglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β-augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N-terminal α-helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. In this issue of Molecular Microbiology, Guérin et al. report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.Molecular Microbiology 05/2014; · 5.03 Impact Factor
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ABSTRACT: Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two-Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI(-) bacteria. CDI(+) bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst α, β, and γ proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5' to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.PLoS Genetics 08/2012; 8(8):e1002877. · 8.52 Impact Factor
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ABSTRACT: The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein Trafficking & Secretion.Biochimica et Biophysica Acta 11/2013; · 4.66 Impact Factor