YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli

The Omp85 family of proteins has been found in all Gram-negative bacteria and even several eukaryotic organisms. The previously uncharacterized Escherichia coli member of this family is YaeT. The results of this study, consistent with previous Omp85 studies, show that the yaeT gene encodes for an essential cellular function. Direct examinations of the outer membrane fraction and protein assembly revealed that cells depleted for YaeT are severely defective in the biogenesis of outer membrane proteins (OMPs). Interestingly, assemblies of the two distinct groups of OMPs that follow either SurA- and lipopolysaccharide-dependent (OmpF/C) or -independent (TolC) folding pathways were affected, suggesting that YaeT may act as a general OMP assembly factor. Depletion of cells for YaeT led to the accumulation of OMPs in the fraction enriched for periplasm, thus indicating that YaeT facilitates the insertion of soluble assembly intermediates from the periplasm to the outer membrane. Our data suggest that YaeT's role in the assembly of OMPs is not mediated through a role in lipid biogenesis, as debated for Omp85 in Neisseria, thus advocating a conserved OMP assembly function of Omp85 homologues.     

Assembly of complex organelles: pilus biogenesis in gram-negative bacteria as a model system

Pathogenic bacteria assemble a variety of adhesive structures on their surface for attachment to host cells. Some of these structures are quite complex. For example, the hair-like organelles known as pili or fimbriae are generally composed of several components and often exhibit composite morphologies. In gram-negative bacteria assembly of pili requires that the subunits cross the cytoplasmic membrane, fold correctly in the periplasm, target to the outer membrane, assemble into an ordered structure, and cross the outer membrane to the cell surface. Thus, pilus biogenesis provides a model for a number of basic biological problems including protein folding, trafficking, secretion, and the ordered assembly of proteins into complex structures. P pilus biogenesis represents one of the best-understood pilus systems. P pili are produced by 80-90% of all pyelonephritic Escherichia coli and are a major virulence determinant for urinary tract infections. Two specialized assembly factors known as the periplasmic chaperone and outer membrane usher are required for P pilus assembly. A chaperone/usher pathway is now known to be required for the biogenesis of more than 30 different adhesive structures in diverse gram-negative pathogenic bacteria. Elucidation of the chaperone/usher pathway was brought about through a powerful combination of molecular, biochemical, and biophysical techniques. This review discusses these approaches as they relate to pilus assembly, with an emphasis on newer techniques.     

Protein secretion and outer membrane assembly in Alphaproteobacteria

The assembly of beta-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of beta-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating beta-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of beta-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, beta-propeller signatures in YfgL). Given that the process of the beta-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of beta-barrel proteins in eukaryotes.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.     

Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.     

Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

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.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

Transport of outer membrane lipids in mycobacteria

The complex organization of the mycobacterial cell wall poses unique challenges for the study of its assembly. Although mycobacteria are classified evolutionarily as Gram-positive bacteria, their cell wall architecture more closely resembles that of Gram-negative organisms. They possess not only an inner cytoplasmic membrane, but also a bilayer outer membrane that encloses an aqueous periplasm and includes diverse lipids that are required for the survival and virulence of pathogenic species. Questions surrounding how mycobacterial outer membrane lipids are transported from where they are made in the cytoplasm to where they function at the cell exterior are thus similar, and similarly compelling, to those that have driven the study of Gram-negative outer membrane transport pathways. However, little is understood about these processes in mycobacteria. Here we contextualize these questions by comparing our current knowledge of mycobacteria with better-defined systems in other organisms. Based on this analysis, we propose possible models and highlight continuing challenges to improving our understanding of outer membrane assembly in these medically and environmentally important bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment

Outer membrane beta-barrel proteins in gram-negative bacteria, such as Escherichia coli, must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different subassembly reactions. This kinetic analysis revealed that secB and secD mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SecD in LamB assembly. Moreover, we found that surA mutants are qualitatively indistinguishable from yfgL mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.     

Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

β-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.     

Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide

YfgL together with NlpB, YfiO, and YaeT form a protein complex to facilitate the insertion of proteins into the outer membrane of Escherichia coli. Without YfgL, the levels of OmpA, OmpF, and LamB are significantly reduced, while OmpC levels are slightly reduced. In contrast, the level of TolC significantly increases in a yfgL mutant. When cells are depleted of YaeT or YfiO, levels of all outer membrane proteins examined, including OmpC and TolC, are severely reduced. Thus, while the assembly pathways of various nonlipoprotein outer membrane proteins may vary through the step involving YfgL, all assembly pathways in Escherichia coli converge at the step involving the YaeT/YfiO complex. The negative effect of yfgL mutation on outer membrane proteins may in part be due to elevated sigma E activity, which has been shown to downregulate the synthesis of various outer membrane proteins while upregulating the synthesis of periplasmic chaperones, foldases, and lipopolysaccharide. The data presented here suggest that the yfgL effect on outer membrane proteins also stems from a defective assembly apparatus, leading to aberrant outer membrane protein assembly, except for TolC, which assembles independent of YfgL. Consistent with this view, the simultaneous absence of YfgL and the major periplasmic protease DegP confers a synthetic lethal phenotype, presumably due to the toxic accumulation of unfolded outer membrane proteins. The results support the hypothesis that TolC and major outer membrane proteins compete for the YaeT/YfiO complex, since mutations that adversely affect synthesis or assembly of major outer membrane proteins lead to elevated TolC levels.     

The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane.

The noncapsid assembly proteins pI and pI of the filamentous bacteriophage f1 are inserted into the inner membrane of Escherichia coli via an internal signal sequence. Inhibition of the activity of SecA with low concentrations of sodium azide results in rapid accumulation of pI and pI proteins in the cytoplasm. However, both proteins are inserted into the membrane under the same conditions when synthesized in bacteria containing a secA azide resistance mutation. The other noncapsid assembly protein, pIV, is an outer membrane protein synthesized with a cleavable signal sequence. Wild-type bacteria accumulate the precursor to pIV when protein synthesis is in the presence of low concentrations of sodium azide. These results suggest that the f1 bacteriophage assembly proteins require SecA and consequently the bacterial Sec system to reach their proper membrane location.     

Complement-mediated inhibition of function in complement-resistant Escherichia coli

C-mediated inhibition of function in C-sensitive strains of Escherichia coli involves the assembly of the membrane attack complex (MAC) on the outer membrane with subsequent inhibition of inner membrane function. The inhibition of inner membrane function is critical for effective cell killing as damage to the outer membrane alone is insufficient to kill a cell in the absence of serum lysozyme. Studies on the measurement of oxygen consumption for cells under complement attack showed that C-sensitive cells were inhibited by assembly of the MAC, and that this represents damage to some component of the respiring inner membrane. Mechanisms of cellular resistance to C attack could include 1) inhibition of the assembly of the MAC, 2) inhibition of effective activation of the assembled MAC, or 3) reversal of the inhibitory effects of the MAC. Demonstration of a transient C-mediated inhibition of inner membrane function in C-resistant cells implies that the latter case should be considered as one possible component of cellular resistance to C attack.     

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

A crosslinking study reveals novel insights into how the chaperone TAM helps Gram-negative bacteria insert the drug efflux pump subunit TolC into their outer membrane. Bioinformatic analyses show that TolC-like proteins can be found in all LPS-containing bacteria, but also in some monodermic Firmicutes.     

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

A mechanism of protein localization: the signal hypothesis and bacteria

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.