Gram-negative bacteria such as Escherichia coli are protected by a complex cell envelope. The development of novel therapeutics against these bacteria necessitates a molecular level understanding of the structure-dynamics-function relationships of the various components of the cell envelope. We use atomistic MD simulations to reveal the details of covalent and noncovalent protein interactions that link the outer membrane to the aqueous periplasmic region. We show that the Braun's lipoprotein tilts and bends, and thereby lifts the cell wall closer to the outer membrane. Both monomers and dimers of the outer membrane porin OmpA can interact with peptidoglycan in the presence of Braun's lipoprotein, but in the absence of the latter, only dimers of OmpA show a propensity to form contacts with peptidoglycan. Our study provides a glimpse of how the molecular components of the bacterial cell envelope interact with each other to mediate cell wall attachment in E. coli.

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Bacteria are protected by complex molecular architectures known as the cell envelope. The cell envelope is composed of regions with distinct chemical compositions and physical properties; namely membranes and a cell wall. To develop novel antibiotics to combat pathogenic bacteria, molecular level knowledge of the structure, dynamics and interplay between the chemical components of the cell envelope that surrounds bacterial cells is imperative. In addition, conserved molecular patterns associated with the bacterial envelope are recognized by receptors as part of the mammalian defensive response to infection, and an improved understanding of bacteria-host interactions would facilitate the search for novel immunotherapeutics. This Perspective introduces an emerging area of computational biology; multiscale molecular dynamics simulations of chemically complex models of bacterial lipids and membranes. We discuss progress to date, and identify areas for future development that will enable the study of aspects of the membrane components that are as yet unexplored by computational methods.Bacteria are divided into two categories, Gramnegative and Gram positive, both of which include pathogens that are harmful to humans. Gram-negative bacteria have cell envelopes composed of two membranes, separated by a region known as the periplasm. The outer membrane (OM) is asymmetric in nature; the two leaflets differ in their compositions. The inner membrane contains a symmetric arrangement of phospholipids. In contrast, Gram-positive bacteria contain only one membrane, which is similar in composition to the inner membrane of Gram-negative bacteria. Both types of bacteria have a cell wall, which is composed of the biopolymer peptidoglycan. The combination of membrane plus cell wall gives rise to the characteristic semi-permeable properties of the cell envelope. To be effective, antibiotics must either cause bacterial cell death or inhibit cell growth. In both cases they must interact with the cell envelope, as they must either (i) disrupt the cell envelope, such that the cell contents leak out, or (ii) cross the cell envelope to gain access to the interior of the cell, where they may interfere with essential cellular process such as DNA replication and metabolism. The emergence of antimicrobial resistance is recognized as a major threat to human health. 1 It is thus imperative to have a detailed knowledge of the structure-dynamics-function relationships of the cell wall and membranes, in order to develop new antibiotics under reduced pressure of resistance. Furthermore, molecules derived from the cell membrane and wall are utilized by the mammalian innate immune system to mount a defensive response. 2 Over-amplification of such pathways can lead to sepsis, which remains the primary cause of death due to infection, highlighting the need for an improved understanding of the molecular mechanisms of immunostimulation. Due to the numerous molecular components involved, studying biological membranes in an in vivo condition rema...

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We present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

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fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes. 1625-Pos Board B534Modeling Colony Pattern Formation under Differential Adhesion and Cell Proliferation Proliferation of individual cells is one of the hallmarks of living systems, and collectively the cells within a colony or tissue form highly structured patterns, influencing the properties at the population level. We develop a cellular automaton model that characterizes bacterial colony patterns emerging from the joint effect of cell proliferation and cell-cell differential adhesion. Through simulations and theoretical analysis akin to interface growth, we show that this model gives rise to novel properties consistent with recent experimental findings. We observe slower than exponential growth in the case of a single cell type as well as new colony patterns in the case of two cell types. In particular, engulfment of one cell type by the other is strongly enhanced compared to the prediction from the equilibrium differential adhesion hypothesis in the absence of proliferation. These observations provide new insights in predicting and characterizing colony morphology using experimentally accessible information such as single cell growth rate and cell adhesion strength. 1626-Pos Board B535 Brauns Lipoprotein Facilitates OmpA Interaction with the Escherichia coliCell Wall Gram-negative bacteria such as Escherichia coli are protected by an extremely complex cellular envelope containing two layers of membranes separated by an aqueous periplasmic space housing a network of cell wall. These membranes are embedded with various proteins that are linked to the cell wall either covalently or non-covalently. Little is currently known on how these interactions are maintained together in such a crowded environment. Here we used atomistic molecular dynamic simulations to reveal the details of simultaneous binding between the outer membrane porin OmpA and the Braun's lipoprotein with the cell wall. Braun's lipoprotein tilts and bends, resulting in a shift of the cell wall towards the outer membrane. This enables both OmpA monomer and dimer to interact with the cell wall. In the absence of Braun's lipoprotein, however, OmpA monomer shows propensity to form contacts with the outer membrane instead. Dimerisation is required to strengthen the electro...