The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli

Fission of bacterial cells involves the co-ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram-negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG-binding OM (lipo)proteins. The Tol-Pal system is energized by proton motive force and is well conserved in Gram-negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein-PG and protein-protein interactions. Although the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells, its precise role has remained unclear. We show that all five components accumulate at constriction sites in Escherichia coli and that mutants lacking an intact system suffer delayed OM invagination and contain large OM blebs at constriction sites and cell poles. We propose that Tol-Pal constitutes a dynamic subcomplex of the division apparatus in Gram-negative bacteria that consumes energy to establish transient trans-envelope connections at/near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction.     

Timing of TolA and TolQ Recruitment at the Septum Depends on the Functionality of the Tol-Pal System

Efficient cell division of Gram-negative bacteria requires the presence of the Tol-Pal system to coordinate outer membrane (OM) invagination with inner membrane invagination (IM) and peptidoglycan (PG) remodeling. The Tol-Pal system is a trans-envelope complex that connects the three layers of the cell envelope through an energy-dependent process. It is composed of the three IM proteins, TolA, TolQ and TolR, the periplasmic protein TolB and the OM lipoprotein Pal. The proteins of the Tol-Pal system are dynamically recruited to the cell septum during cell division. TolA, the central hub of the Tol-Pal system, has three domains: a transmembrane helix (TolA₁), a long second helical periplasmic domain (TolA₂) and a C-terminal globular domain (TolA₃). The TolQR complex uses the PMF to energize TolA, allowing its cyclic interaction via TolA₃ with the OM TolB-Pal complex. Here, we confirm that TolA₂ is sufficient to address TolA to the site of constriction, whereas TolA₁ is recruited by TolQ. Analysis of the protein localization as function of the bacterial cell age revealed that TolA and TolQ localize earlier at midcell in the absence of the other Tol-Pal proteins. These data suggest that TolA and TolQ are delayed from their septal recruitment by the multiple interactions of TolA with TolB-Pal in the cell envelope providing a new example of temporal regulation of proteins recruitment at the septum.     

Membrane localization of motility,signaling, and polyketide synthetase proteins in Myxococcus xanthus

Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm(-3)). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm(-3)) and low buoyant density (1.169 to 1.171 g cm(-3)). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm(-3)) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.     

Biogenesis of bacterial membrane vesicles

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo . Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein–peptidoglycan (OM–PG) and OM protein–inner membrane protein (OM–PG–IM) interactions within the envelope structure. Modulation of OM–PG and OM–PG–IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM–PG and OM–PG–IM associations within the envelope structure, thus releasing OM as MVs.     

New insights into the Lpt machinery for lipopolysaccharide transport to the cell surface: LptA-LptC interaction and LptA stability as sensors of a properly assembled transenvelope complex

Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport.     

Changes of mitochondrial membrane proteins in rat cerebellum during aging

Qualitative and quantitative changes of mitochondrial membrane proteind during aging were investigated. Free (non-synaptic) mitochondria were purified from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months). Mitochondrial outer membrane (OM), inner membrane (IM) and matrix (MX) were separated and the proteins were extracted and analyzed by gel-electrophoresis.After staining, the gels were scanned densitometrically to quantify the proteins. No significant changes in the quantity of OM or MX protein subunits were observed, while serveral statistically significant quantitative changes in IM proteins with age were found. These age-dependent modifications of inner membrane mitochondrial proteins may play an important role in energy transduction, transport systems and regulatory enzymatic activities in mitochondria.     

Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.     

How complement kills E. coli. I. Location of the lethal lesion

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.     

Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic sigma factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.     

Absence of YhdP,TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria provides the cell with a formidable barrier that excludes external threats. The two major constituents of this asymmetric barrier are lipopolysaccharide (LPS) found in the outer leaflet, and glycerophospholipids (GPLs) in the inner leaflet. Maintaining the asymmetric nature and balance of LPS to GPLs in the OM is critical for bacterial viability. The biosynthetic pathways of LPS and GPLs are well characterized, but unlike LPS transport, how GPLs are translocated to the OM remains enigmatic. Understanding this aspect of cell envelope biology could provide a foundation for new antibacterial therapies. Here, we report that YhdP and its homologues, TamB and YdbH, members of the “AsmA-like” family, are critical for OM integrity and necessary for proper GPL transport to the OM. The absence of the two largest AsmA-like proteins (YhdP and TamB) leads to cell lysis and antibiotic sensitivity, phenotypes that are rescued by reducing LPS synthesis. We also find that yhdP, tamB double mutants shed excess LPS through outer membrane vesicles, presumably to maintain OM homeostasis when normal anterograde GPL transport is disrupted. Moreover, a yhdP, tamB, ydbH triple mutant is synthetically lethal, but if GPL transport is partially restored by overexpression of YhdP, the cell shape adjusts to accommodate increased membrane content as the cell accumulates GPLs in the IM. Our results therefore suggest a model in which “AsmA-like” proteins transport GPLs to the OM, and when hindered, changes in cell shape and shedding of excess LPS aids in maintaining OM asymmetry.     

Structural insight into lipopolysaccharide transport from the Gram-negative bacterial inner membrane to the outer membrane

Lipopolysaccharide (LPS) is an important component of the outer membrane (OM) of Gram-negative bacteria, playing essential roles in protecting bacteria from harsh environments, in drug resistance and in pathogenesis. LPS is synthesized in the cytoplasm and translocated to the periplasmic side of the inner membrane (IM), where it matures. Seven lipopolysaccharide transport proteins, LptA-G, form a trans‑envelope complex that is responsible for LPS extraction from the IM and transporting it across the periplasm to the OM. The LptD/E of the complex transports LPS across the OM and inserts it into the outer leaflet of the OM. In this review we focus upon structural and mechanistic studies of LPS transport proteins, with a particular focus upon the LPS ABC transporter LptB₂FG. This ATP binding cassette transporter complex consists of twelve transmembrane segments and has a unique mechanism whereby it extracts LPS from the periplasmic face of the IM through a pair of lateral gates and then powers trans‑periplasmic transport to the OM through a slide formed by either of the periplasmic domains of LptF or LptG, LptC, LptA and the N-terminal domain of LptD. The structural and functional studies of the seven lipopolysaccharide transport proteins provide a platform to explore the unusual mechanisms of LPS extraction, transport and insertion from the inner membrane to the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Distribution of mechanical stress in the Escherichia coli cell envelope

The cell envelope in Gram-negative bacteria comprises two distinct membranes with a cell wall between them. There has been a growing interest in understanding the mechanical adaptation of this cell envelope to the osmotic pressure (or turgor pressure), which is generated by the difference in the concentration of solutes between the cytoplasm and the external environment. However, it remains unexplored how the cell wall, the inner membrane (IM), and the outer membrane (OM) effectively protect the cell from this pressure by bearing the resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis. In this study, we have used molecular dynamics (MD) simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension. We calculated the area compressibility modulus of all three components in simulations from tension-area isotherms. Experiments on monolayers mimicking individual leaflets of the IM and OM were also used to characterize their compressibility. While the membranes become softer as they expand, the cell wall exhibits significant strain stiffening at moderate to high tensions. We integrate these results into a model of the cell envelope in which the OM and cell wall share the tension at low turgor pressure (0.3 atm) but the tension in the cell wall dominates at high values (>1 atm).     

The lipopolysaccharide transport system of Gram-negative bacteria

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex lipid with important biological activities. In the host it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM. The chemical structure of LPS and its biosynthetic pathways have been fully elucidated. By contrast only recently details of the transport and assembly of LPS into the OM have emerged. LPS is synthesized in the cytoplasm and at the inner leaflet of the IM and needs to cross two different compartments, the IM and the periplasm, to reach its final destination at the OM. This review focuses on recent studies that led to our present understanding of the protein machine implicated in LPS transport and in assembly at the cell surface.     

Outside-In Assembly Pathway of the Type IV Pilus System in Myxococcus xanthus

Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.     

Characterization of lipopolysaccharide transport protein complex

Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.     

Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.     

Identification and localization of Myxococcus xanthus porins and lipoproteins

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.     

Deciphering the assembly of the Yersinia type III secretion injectisome

The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer‐membrane (OM) ring), YscD (forms the inner‐membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside‐in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co‐immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.