A Prokaryotic Membrane Sculpting BAR Domain Protein

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here we report the first prokaryotic BAR domain protein, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and scaffolding OMEs into a consistent diameter and curvature. Cryogenic transmission electron microscopy reveals a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubes produced by the wild type strain. Overexpression of BdpA promotes OME formation during conditions where they are less common. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane curvature in vivo, we propose that BdpA and its homologs comprise a newly identified class of prokaryotic BAR (P-BAR) domains

In Situ Imaging of Bacterial Outer Membrane Projections and Associated Protein Complexes Using Electron Cryo-Tomography

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor

Geobacter sulfurreducens Extracellular Multiheme Cytochrome PgcA Facilitates Respiration to Fe(III) Oxides But Not Electrodes

Extracellular cytochromes are hypothesized to facilitate the final steps of electron transfer between the outer membrane of the metal-reducing bacterium Geobacter sulfurreducens and solid-phase electron acceptors such as metal oxides and electrode surfaces during the course of respiration. The triheme c-type cytochrome PgcA exists in the extracellular space of G. sulfurreducens, and is one of many multiheme c-type cytochromes known to be loosely bound to the bacterial outer surface. Deletion of pgcA using a markerless method resulted in mutants unable to transfer electrons to Fe(III) and Mn(IV) oxides; yet the same mutants maintained the ability to respire to electrode surfaces and soluble Fe(III) citrate. When expressed and purified from Shewanella oneidensis, PgcA demonstrated a primarily alpha helical structure, three bound hemes, and was processed into a shorter 41 kDa form lacking the lipodomain. Purified PgcA bound Fe(III) oxides, but not magnetite, and when PgcA was added to cell suspensions of G. sulfurreducens, PgcA accelerated Fe(III) reduction similar to addition of FMN. Addition of soluble PgcA to ΔpgcA mutants also restored Fe(III) reduction. This report highlights a distinction between proteins involved in extracellular electron transfer to metal oxides and poised electrodes, and suggests a specific role for PgcA in facilitating electron transfer at mineral surfaces