Biology subjects

Silale, A.

Publications and source records attributed to Silale, A..

4 recordsLinked to original sources

bioRxiv2022

BtuB TonB-dependent transporters and BtuG surface lipoproteins form stable complexes for vitamin B12 uptake in gut Bacteroides.

Vitamin B12 (cobalamin) is the most complex vitamin and essential for many human gut microbes. However, cobalamin is synthesised only by a limited number of bacteria, making many gut microbes dependent on scavenging to meet their cobalamin requirements. Since bacterial densities in the gut are extremely high, competition for cobalamin is severe, making it a keystone micronutrient that shapes human gut microbial communities. Contrasting with Enterobacteria like Escherichia coli which only have one outer membrane (OM) transporter dedicated to B12 uptake (BtuB), members of the dominant genus Bacteroides often encode several vitamin B12 OM transporters together with a conserved array of surface-exposed B12-binding lipoproteins. Here we show, via X-ray crystallography, cryogenic electron microscopy (cryoEM) and molecular dynamics (MD) simulations, that the BtuB1 and BtuB2 transporters from the prominent human gut bacterium Bacteroides thetaiotaomicron form stable complexes with the surface-exposed lipoproteins BtuG1 and BtuG2. The lipoproteins cap the external surface of their cognate BtuB transporter and, when open, capture B12 via electrostatic attraction. After B12 capture, the BtuG lid closes, with concomitant transfer of the vitamin to the BtuB transporter and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut.

microbiology↗

bioRxiv2022

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Bacteroidetes are abundant members of the human microbiota, with species occupying the distal gut capable of utilising a myriad of diet- and host-derived glycans. Transport of glycans across the outer membrane (OM) of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, that are thought to operate via a pedal-bin mechanism in which the lids open and close to facilitate substrate binding. However, additional cell surface-exposed lipoproteins, namely surface glycan binding proteins and glycoside hydrolases, play critical roles in the capture and processing of large glycan chains into transport-competent substrates. Despite constituting a crucial mechanism of nutrient acquisition by our colonic microbiota, the interactions between these components in the OM are poorly understood. Here we show that for the levan and dextran utilisation systems of Bacteroides thetaiotaomicron, the additional OM components assemble on the core SusCD transporter, forming stable glycan utilising machines which we term utilisomes. Single particle electron cryogenic electron microscopy (cryo-EM) structures in the absence and presence of substrate reveal concerted conformational changes that rationalise the role of each component for efficient nutrient capture, as well as providing a direct demonstration of the pedal bin mechanism of substrate capture in the intact utilisome.

microbiology↗

bioRxiv2022

Structural basis for host recognition and superinfection exclusion by bacteriophage T5

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent a secondary viral infection by the same or a closely related phage, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here we present the cryo-EM structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug, but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a non-native conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of E. coli by phage T5, and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

microbiology↗

bioRxiv2020

Structure of a proton-powered molecular motor that drives protein transport and gliding motility

Ion-driven motors are rare in biology. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Whilst the molecular mechanism of ATP synthase is now well understood, structural information is lacking for the other two classes of motor. Here we present the structure of the Bacteroidetes gliding motility motor determined by cryo-electron microscopy. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane. This work provides a molecular basis for understanding how the gliding motility motor is able to transduce the energy of the inner membrane protonmotive force across the bacterial cell envelope.

biochemistry↗