Dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes.

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group have recently been shown to lack well-characterised OM attachment systems, but instead have OmpM, which could represent an ancestral tethering system in bacteria. Here, we have determined the structure of the most abundant OmpM protein from Veillonella parvula (diderm Firmicutes) by single particle cryogenic electron microscopy. We also characterised the channel properties of the transmembrane ß-barrel of OmpM and investigated the structure and PG-binding properties of its periplasmic stalk region. Our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula, and probably other diderm Terrabacteria. This dual function of OmpM may have played a role in the loss of the OM in ancestral bacteria and the emergence of monoderm bacterial lineages.

Computational microbiology of bacteria: Advancements in molecular dynamics simulations.

Microbiology is traditionally considered within the context of wet laboratory methodologies. Computational techniques have a great potential to contribute to microbiology. Here, we describe our loose definition of "computational microbiology" and provide a short survey focused on molecular dynamics simulations of bacterial systems that fall within this definition. It is our contention that increased compositional complexity and realistic levels of molecular crowding within simulated systems are key for bridging the divide between experimental and computational microbiology.

A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer.

Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism are largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified, and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens.

The fatty membrane that surrounds cells is an essential feature of all living things. It is a selective barrier, only allowing certain substances to enter and exit the cell, and it contains the proteins and carbohydrates that the cell uses to interact with its environment. In bacteria, the carbohydrates on the outer side of the membrane can become 'tagged' or modified with small chemical entities which often prove useful for the cell. Acyl groups, for example, allow disease-causing bacteria to evade the immune system and contribute to infections persisting in the body. As a rule, activated acyl groups are only found inside the cell, so they need to move across the membrane before they can be attached onto the carbohydrates at the surface. This transfer is performed by a group of proteins that sit within the membrane called the acyltransferase-3 (AT3) family. The structure of these proteins and the mechanism by which they facilitate membrane crossing have remained unclear. Newman, Tindall et al. combined computational and structural modelling techniques with existing experimental data to establish how this family of proteins moves acyl groups across the membrane. They focused on OafB, an AT3 protein from the foodborne bacterial pathogen Salmonella typhimurium. The experimental data used by the team included information about which parts of OafB are necessary for this protein to acylate carbohydrates molecules. In their experiments, Newman, Tindall et al. studied how different parts of OafB move, how they interact with the molecules that carry an acyl group to the membrane, and how the acyl group is then transferred to the carbohydrate acceptor. Their results suggest that AT3 family proteins have a central pore or hole, plugged by a loop. This loop moves and therefore 'unplug' the pore, resulting in the emergence of a channel across the membrane. This channel can accommodate the acyl-donating molecule, presenting the acyl group to the outer surface of the membrane where it can be transferred to the acceptor carbohydrate. The AT3 family of proteins participates in many cellular processes involving the membrane, and a range of bacterial pathogens rely on these proteins to successfully infect human hosts. The results of Newman Tindall et al. could therefore be used across the biological sciences to provide more detailed understanding of the membrane, and to inform the design of drugs to fight bacterial diseases.

Conformational dynamics and putative substrate extrusion pathways of the N-glycosylated outer membrane factor CmeC from Campylobacter jejuni.

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the protein is N-glycosylated, with the glycans playing a key role in the effective function of this system. In this work we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid and a macrolide antibiotic. These, and additional equilibrium simulations suggest that the anionic bile acid utilises multivalent cations to climb the ladder of acidic residues that line the interior surface of the protein.