Braun's Lipoprotein Research Articles

Controllable Autolytic Leaky E. coli Platform for the Recovery of Intracellular Proteins.

Escherichia coli is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic E. coli strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of lpp (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.

Overexpression of l,d-Transpeptidase A Induces Dispensability of Rod Complex in Escherichia coli.

Antimicrobial resistance (AMR) is a significant global threat, and the presence of resistance-determinant genes is one of the major driving forces behind it. The bacterial rod complex is an essential set of proteins that is crucial for cell survival due to its role in cell wall biogenesis and shape maintenance. Therefore, these proteins offer excellent potential as drug targets; however, compensatory mutations in nontarget genes render this complex nonessential. The MreB protein of this complex is an actin homologue that rotates along the longitudinal axis of the cell to provide rod shape to the bacteria. In this study, using chemical-chemical interaction profiling and FtsZ suppression assay, we identified the MreB targeting activity of IITR07865, a previously discovered small molecule in our lab. Escherichia coli suppressors against IITR07865 revealed mutations in two cell division-associated genes, min C and pal, that have not been previously implicated in rod complex essentiality. IITR07865 resistant mutants were found to inactivate and render the rod complex nonessential, making the rod complex inhibitors ineffective. Further, through transcriptome analysis, we reveal the primary cause of resistance in suppressor strains to be the overexpression of an l, d-transpeptidase A enzyme, which is involved in peptidoglycan and Braun's lipoprotein cross-linking. Our results demonstrate a novel mechanism of resistance development in rod-shaped Gram-negative bacterial pathogen E. coli involved in UTIs where mecillinam, a clinically used antibiotic that targets rod complex, is a drug of choice.

Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S.vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18°C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S.vesiculosa HM13 as a host.

Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.

Molecular Crowding Alters the Interactions of Polymyxin Lipopeptides within the Periplasm of E. coli: Insights from Molecular Dynamics.

The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the E. coli periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.

Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun's lipoprotein from peptidoglycan.

Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

Prostaglandin D2 regulates Escherichia coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages

Prostaglandin D2 (PGD2) synthesis is closely associated with the innate immune response mediated by pattern recognition receptors (PPRs). We determined PGD2 synthesis whether mediated by Toll-like receptor 2 (TLR2), TLR4 and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) in Escherichia coli (E. coli)-, lipopolysaccharide (LPS)- and Braun lipoprotein (BLP)-stimulated macrophages. Our data demonstrate that TLR2, TLR4, and NLRP3 could regulate the synthesis of PGD2 through cyclo-oxygenase-2 (COX-2) and hematopoietic PGD synthase (H-PGDS) in E. coli-, LPS- or BLP-stimulated macrophages, suggesting that TLR2, TLR4, and NLRP3 are critical in regulating PGD2 secretion by controlling PGD2 synthetase expression in E. coli-, LPS- or BLP-stimulated macrophages. The H-PGDS (a PGD2 specific synthase) inhibitor pre-treatment could down-regulate the secretion of TNF-α, RANTES and IL-10 in LPS- and E. coli-stimulated macrophage. Meanwhile, H-PGDS inhibitor could down-regulate the secretion of TNF-α, while up-regulated RANTES and IL-10 secretion in BLP-stimulated macrophages, suggesting that PGD2 could regulate the secretion of cytokines and chemokines in E. coli-, LPS- or BLP-stimulated macrophages. Furthermore, exogenous PGD2 regulates the secretion of cytokines and chemokines through activation of MAPK and NF-κB signaling pathways after E. coli-, LPS- or BLP stimulation in macrophages. Taken together, PGD2 is found able to regulate E. coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages.

Braun Lipoprotein Protects against Escherichia coli-Induced Inflammatory Responses and Lethality in Mice

Escherichia coli (E. coli), a Gram-negative bacterium, is an important pathogen that causes several mammalian diseases. The outer membrane components of E. coli, namely, lipopolysaccharide (LPS) and bacterial lipoprotein, can induce the host innate immune response through pattern recognition receptors (PRRs). However, the detailed roles of the E. coli Braun lipoprotein (BLP) in the regulation of host inflammatory response to E. coli infection remain unclear. In this study, we sought to determine the effects of BLP on E. coli-induced host inflammatory response and lethality using mouse models. Experiments using the E. coli DH5α strain (BLP-positive), E. coli JE5505 strain (BLP-negative), and E. coli JE5505 strain combined with BLP indicated that the presence of BLP could alleviate mortality and organ (liver and lung) damage and decrease proinflammatory cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β]) and chemokine (regulated on activation normal T-cell expressed and secreted [RANTES]) production in mouse serum and organs. Conversely, E. coli JE5505, E. coli DH5α strain, and E. coli JE5505 combined with BLP treatment induce enhanced anti-inflammatory cytokine (interleukin 10 [IL-10]) production in mouse serum and organs. In addition, BLP could regulate the secretion of proinflammatory cytokines (TNF-α and IL-1β), chemokines (RANTES), and anti-inflammatory factors (IL-10) through mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling pathways in macrophages. Altogether, our results demonstrate that the bacterial component BLP plays crucial and protective roles in E. coli-infected mice, which may influence the outcome of inflammation in host response to E. coli infection. IMPORTANCE In this study, we investigated the roles of bacterial outer membrane component BLP in regulating inflammatory responses and lethality in mice that were induced by a ubiquitous and serious pathogen, Escherichia coli. BLP could alleviate the mortality of mice and organ damage, as well as decrease proinflammatory cytokines and chemokine production and enhance anti-inflammatory cytokine production in mouse serum and organs. Overall, our results demonstrate that the bacterial component BLP plays crucial and protective roles in E. coli-infected mice through regulating the production of an inflammatory mediator, which may influence the outcome of inflammation in host response to E. coli infection. Our findings provide new information about the basic biology involved in immune responses to E. coli and host-bacterial interactions, which have the potential to translate into novel approaches for the diagnosis and treatment of E. coli-related medical conditions, such as bacteremia and sepsis.

Complexity matters: Polymyxins and the E. coli cell envelope.

We present a computational study of the effects of biomolecular crowding on the behaviour of the antimicrobial peptides (AMPs) polymyxin B1 (PMB) and polymyxin E (PME) within the periplasm of E. coli. We have employed atomistic molecular dynamics simulations to explore the nature of the interaction between these AMPs and both Braun's lipoprotein (BLP) and the peptidoglycan (PGN) cell wall in models of the periplasm that are crowded to different extents. The most crowded model of the periplasm contained a range of osmolytes as well as crowding proteins (ubiquitin) in addition to the peptides, cell wall and Braun's lipoprotein. All systems also included a model of the E. coli outer membrane. Overall, three x 250 ns replicates of each system were performed (with both just neutralising ions and also 150 mMol concentration of NaCl); giving a total simulation time of 10.5 microseconds. Our results show the importance of D-phenylalanine to the primarily hydrophobic interaction between PMB and BLP; we characterise the change in this interaction with increased molecular crowding and highlight how the replacement of this moiety by D-leucine in PME altered the balance of interactions. A similar analysis of the interactions between the peptides and the cell wall highlight the prevalence of electrostatic interactions between the specific (DAP) residues of the cell wall and both polymyxins. Finally, the impacts of both polymyxins on the dimensions of the ‘mesh’ of the cell wall are discussed. Our results highlight the importance of considering the full biological complexity of a system when attempting to characterise details about the function and behaviour of antimicrobial peptides in vivo; as evidenced by an increase in the range of observed behavioural states of polymyxins as the complexity of the system increased.

Thicket and mesh: Understanding turgor pressure resistance of outer membrane-cell wall system.

The cell envelope of Gram-negative bacteria is composed of three layers: the outer membrane (OM), the peptidoglycan cell wall (PG), and the inner membrane. Together, the cell envelope provides a physical and chemical barrier that protects against the turgor pressure generated by the solute concentration difference between the inside and outside of bacteria, which can range from 0.2 atm to 5 atm. Previously, we measured the contribution of each component to see how it responds to different strains and determined that most of the cell envelope's strength comes from the OM and PG. However, the combined contribution from the OM and the PG, especially considering the Braun's lipoprotein (Lpp) that connects the two, remains to be elucidated. To address this, we constructed and simulated a combined OM-PG system that contains both OM and PG connected by Lpp. We also imposed different initial PG strain levels by stretching it by 2.0X and 2.5X its original size. The results showed that OM can resist imposed tensions by PG up to 2.0x its resting size. Moreover, we explored how the OM/PG-spanning protein TolC can affect the overall resistance to tension, through both enhancement of the connections between the layers and weakening them individually.

An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition.

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun's lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Cleavage of Braun’s lipoprotein Lpp from the bacterial peptidoglycan by a paralog of l,d-transpeptidases, LdtF

The gram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun's lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG-Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly identified paralog of l,d-transpeptidases in E. coli, is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG-Lpp cross-links, suggesting a role for LdtF in the regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.

β-Barrel proteins tether the outer membrane in many Gram-negative bacteria.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In E. coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4 but most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We use a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in stationary phase and tethering of the lipoprotein LimB similar throughout the cell-cycle. Using a genetic approach, we demonstrate that the cell-cycle dependent tethering of BbpA is partly dependent on a developmentally regulated L,D transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell-cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

β-Barrels covalently link peptidoglycan and the outer membrane in the α-proteobacterium Brucella abortus.

Gram-negative bacteria are surrounded by a cell envelope that comprises an outer membrane (OM) and an inner membrane that, together, delimit the periplasmic space, which contains the peptidoglycan (PG) sacculus. Covalent anchoring of the OM to the PG is crucial for envelope integrity in Escherichia coli. When the OM is not attached to the PG, the OM forms blebs and detaches from the cell. The Braun lipoprotein Lpp1 covalently attaches OM to the PG but is present in only a small number of γ-proteobacteria; the mechanism of OM-PG attachment in other species is unclear. Here, we report that the OM is attached to PG by covalent cross-links between the N termini of integral OM β-barrel-shaped proteins (OMPs) and the peptide stems of PG in the α-proteobacteria Brucella abortus and Agrobacterium tumefaciens. Cross-linking is catalysed by L,D-transpeptidases and attached OMPs have a conserved alanyl-aspartyl motif at their N terminus. Mutation of the aspartate in this motif prevents OMP cross-linking and results in OM membrane instability. The alanyl-aspartyl motif is conserved in OMPs from Rhizobiales; it is therefore feasible that OMP-PG cross-links are widespread in α-proteobacteria.

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.

Acute phase protein, α – 1- acid glycoprotein (AGP-1), has differential effects on TLR-2 and TLR-4 mediated responses

Alpha-1-acid glycoprotein (AGP-1) is a major positive acute phase glycoprotein with unknown functions that likely play a role in inflammation. We tested its involvement in a variety of inflammatory responses using human AGP-1 purified to apparent homogeneity and confirmed its identity by immunoblotting and mass spectrometry. AGP-1 alone upregulated MAPK signaling in murine peritoneal macrophages. However, when given in combination with TLR ligands, AGP-1 selectively augmented MAPK activation induced by ligands of TLR-2 (Braun lipoprotein) but not TLR-4 (lipopolysaccharide). In vivo treatment of AGP-1 in a murine model of sepsis with or without TLR-2 or TLR-4 ligands, selectively potentiated TLR-2-mediated mortality, but was without significant effect on TLR-4-mediated mortality. Furthermore, in vitro, AGP-1 selectively potentiated TLR-2 mediated adhesion of human primary immune cell, neutrophils. Hence, our studies highlight a new role for the acute phase protein AGP-1 in sepsis via its interaction with TLR-2 signaling mechanisms to selectively promote responsiveness to one of the two major gram-negative endotoxins, contributing to the complicated pathobiology of sepsis.

Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain the Local Structure of the E. coli Cell Wall

We present a molecular modeling and simulation study 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. 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. 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.

Atomistic and Coarse Grain Simulations of the Cell Envelope of Gram-Negative Bacteria: What Have We Learned?

Bacterial membranes, and those of Gram-negative bacteria in particular, are some of the most biochemically diverse membranes known. They incorporate a wide range of lipid types and proteins of varying sizes, architectures, and functions. While simpler biological membranes have been the focus of myriad simulation studies over the years that have yielded invaluable details to complement, and often to direct, ongoing experimental studies, simulations of complex bacterial membranes have been slower to emerge. However, the past few years have seen tremendous activity in this area, leading to advances such as the development of atomistic and coarse-grain models of the lipopolysaccharide (LPS) component of the outer membrane that are compatible with widely used simulation codes. In this Account, we review our contributions to the field of molecular simulations of the bacterial cell envelope, including the development of models of both membranes and the cell wall of Gram-negative bacteria, with a predominant focus on E. coli. At the atomistic level, simulations of chemically accurate models of both membranes have revealed the tightly cross-linked nature of the LPS headgroups and have shown that penetration of solutes through these regions is not as straightforward as the route through phospholipids. The energetic differences between the two routes have been calculated. Simulations of native outer membrane proteins in LPS-containing membranes have shown that the conformational dynamics of the proteins is not only slower in LPS but also different compared to in simpler models of phospholipid bilayers. These chemically more complex and consequently biologically more relevant models are leading to details of conformational dynamics that were previously inaccessible from simulations. Coarse-grain models have enabled simulations of multiprotein systems on time scales of microseconds, leading to insights not only into the rates of protein and lipid diffusion but also into the trends in their respective directions of flow. We find that the motions of LPS molecules are highly correlated with each other but also with outer membrane proteins embedded within the membrane. We have shown that the two leaflets of the outer membrane exhibit communication, whereby regions of low disorder in one leaflet correspond to regions of high disorder in the other. The cell wall remains a comparatively neglected component, although models of the E. coli peptidoglycan are now emerging, particularly at the atomistic level. Our simulations of Braun's lipoprotein have shown that bending and tilting of this protein afford a degree of variability in the gap between the cell wall and the OM. The noncovalent interactions with the cell wall of proteins such as OmpA can further influence the width of this gap by extension or contraction of their linker domains. Overall we have shown that the dynamics of proteins, lipids, and other molecular species within the outer membrane cannot be approximated using simpler phospholipid bilayers, if one is addressing questions regarding the in vivo behavior of Gram-negative bacteria. These membranes have their own unique chemical characteristics that cannot be decoupled from their biological functions.