The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded β-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal β-barrel domain.

β-barrel membrane proteins fold via hybrid-barrel intermediate states.

Gram-negative bacteria and eukaryotic organelles of bacterial origin contain outer membrane proteins that possess a transmembrane "β-barrel" domain. The conserved β-barrel assembly machine (BAM) and the sorting and assembly machine (SAM) are required for the folding and membrane insertion of β-barrels in Gram-negative bacteria and mitochondria, respectively. Although the mechanisms by which β-barrels are folded are incompletely understood, advances in cryo-electron microscopy (cryo-EM) have recently yielded unprecedented insights into their folding process.

Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the β-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods.

In Vivo Disulfide-Bond Crosslinking to Study β-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded β-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of β-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of β-barrels in situ.

The patatin-like protein PlpD forms novel structurally dynamic homodimers in the outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded β-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal β-barrel domain.

Small Molecule Antibiotics Inhibit Distinct Stages of Bacterial Outer Membrane Protein Assembly.

Several antibacterial compounds have recently been discovered that potentially inhibit the activity of BamA, an essential subunit of a heterooligomer (the arrel ssembly achinery or BAM) that assembles outer membrane proteins (OMPs) in Gram-negative bacteria, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors on the biogenesis of a model E. coli OMP (EspP) .

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of β-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies.

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding.

BamA forms a translocation channel for polypeptide export across the bacterial outer membrane.

The β-barrel assembly machine (BAM) integrates β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own β-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of β-barrel proteins, BamA functions as a polypeptide export channel.

The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s).

Unprecedented Abundance of Protein Tyrosine Phosphorylation Modulates Shigella flexneri Virulence.

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri.

A small conserved motif supports polarity augmentation of Shigella flexneri IcsA.

The rod-shaped enteric intracellular pathogen Shigella flexneri and other Shigella species are the causative agents of bacillary dysentery. S. flexneri are able to spread within the epithelial lining of the gut, resulting in lesion formation, cramps and bloody stools.

The passenger-associated transport repeat promotes virulence factor secretion efficiency and delineates a distinct autotransporter subtype.

Autotransporters are a superfamily of virulence factors secreted by Gram negative bacteria. They are comprised of an N-terminal passenger domain that is translocated across the outer membrane and a C-terminal domain that inserts into the outer membrane forming a β-barrel anchor. It is still poorly understood how the passenger is efficiently translocated in the absence of external energy inputs.

Lipopolysaccharide surface structure does not influence IcsA polarity.

Shigella species are the causative agents of human bacillary dysentery. These bacteria spread within the lining of the gut via a process termed actin-based motility whereby an actin 'tail' is formed at the bacterial pole. The bacterial outer membrane protein IcsA initiates this process, and crucially is precisely positioned on the bacterial polar surface.

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles.

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised.