Characterization of pores formed by YaeT (Omp85) from Escherichia coli

Proteins of the Omp85 family play a major role in the biogenesis of the bacterial outer membrane, since they were shown to mediate insertion of outer membrane proteins. The Escherichia coli Omp85 homologue YaeT is essential for viability, but its exact mode of action is not yet elucidated. We could show that YaeT is composed of two distinct domains, an amino-terminal periplasmic and a carboxy-terminal membrane domain. The full length YaeT and the isolated membrane domain induce pores when reconstituted in planar lipid membranes. The pores exhibit a certain variability of conductance indicating a flexible structure, which could be an essential property of a lateral opening channel releasing proteins into the bacterial outer membrane. We could further show that the periplasmic domain proves to be essential for in vivo function of YaeT.     

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85

β‐barrel‐shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell‐wall of Gram‐negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane‐localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N‐terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline‐rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.     

Characterisation of YtfM, a second member of the Omp85 family in Escherichia coli

Omp85 proteins form a ubiquitous protein family, members of which are found in all Gram-negative bacteria. Omp85 of Neisseria meningitidis and YaeT of Escherichia coli are shown to be essential for outer membrane biogenesis. Interestingly, there exists a homologue to YaeT in E. coli and many proteobacteria, denoted YtfM, the function of which has not been described yet. Like YaeT, YtfM is predicted to consist of an amino-terminal periplasmic domain and a membrane-located carboxy-terminal domain. In this study, we present a first characterisation of YtfM by comparison to YaeT concerning structural, biochemical and electrophysiological properties. Furthermore, a knockout strain revealed that ytfM is a non-essential gene and lack of the protein had no effect on outer membrane composition and integrity. The only observable phenotype was strongly reduced growth, indicating an important role of YtfM in vivo.     

YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli

Recent advances in the study of bacterial membranes have led to the identification of a multicomponent YaeT complex in the outer membrane (OM) of Gram-negative bacteria that is involved in the targeting and folding of beta-barrel outer membrane proteins (OMPs). In Escherichia coli, this complex consists of an essential OMP, YaeT, and three OM lipoproteins, YfgL, NlpB and YfiO. YfiO is the only essential lipoprotein component of the complex. We show that this lipoprotein is required for the proper assembly and/or targeting of OMPs to the OM but not the assembly of lipopolysaccharides (LPS). Depletion of YfiO causes similar phenotypes as does the depletion of YaeT, and we conclude that YfiO plays a critical role in YaeT-mediated OMP folding. We demonstrate that YfiO and YfgL directly interact with YaeT in vitro, while NlpB interacts directly with YfiO. Genetic analysis verifies the importance of YfiO and its interactions with NlpB in maintaining the functional integrity of the YaeT complex.     

Interactions between folding factors and bacterial outer membrane proteins

The outer membrane is the first line of contact between Gram-negative bacteria and their external environment. Embedded in the outer membrane are integral outer membrane proteins (OMPs) that perform a diverse range of tasks. OMPs are synthesized in the cytoplasm and are translocated across the inner membrane and probably diffuse through the periplasm before they are inserted into the outer membrane in a folded and biologically active form. Passage through the periplasm presents a number of challenges, due to the hydrophobic nature of the OMPs and the choice of membranes into which they can insert. Recently, a number of periplasmic proteins and one OMP have been shown to play a role in OMP biogenesis. In this review, we describe what is known about these folding factors and how they function in a biological context. In particular, we focus on how they interact with the OMPs at the molecular level and present a comprehensive overview of data relating to a possible effect on OMP folding yield and kinetics. Furthermore, we discuss the role of lipo-chaperones, i.e. lipopolysaccharide and phospholipids, in OMP folding. Important advances have clearly been made in the field, but much work remains to be done, particularly in terms of describing the biophysical basis for the chaperone-OMP interactions which so intricately regulate OMP biogenesis.     

Crystal Structure of a Major Outer Membrane Protein from Thermus thermophilus HB27

The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new β-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 Å, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded β-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular β-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this β-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.     

Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of β-Barrel Outer Membrane Proteins, Including That of BamA Itself

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.     

First glimpse of the crystal structure of YaeT's POTRA domains.

The Omp85/YaeT family of proteins, which are conserved from bacteria to human, catalyzes insertion and assembly of proteins in the outer membrane. The structure consists of a transmembrane beta-barrel domain and a soluble polypeptide-transport-associated (POTRA) domain. The POTRA domain is critical for substrate recognition and perhaps substrate folding, while the beta-barrel domain assists in membrane insertion. The resolution of the crystal structure of the POTRA domain of the Escherichia coli YaeT protein provides a possible molecular mechanism by which the diverse group of substrates is recognized. Knowledge gained from the crystal structure may also spur the development of a novel class of chemotherapeutic inhibitors.     

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.     

Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 degrees C. After 30 min at 44 degrees C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 degrees C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.     

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.     

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.     

The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.Membrane-embedded β-barrel proteins are found in the outer membranes (OMs) of gram-negative bacteria, mitochondria, and chloroplasts. Only in recent years have cellular components required for the assembly and insertion of these OM proteins (OMPs) into the OM been identified. Omp85, which was first characterized in Neisseria meningitidis, is the key protein of the OMP assembly machinery (41). The function of Omp85 has been preserved during evolution, not only in gram-negative bacteria (8, 37, 44, 46) but also in mitochondria, where an Omp85 homolog, also known as Tob55 or Sam50, was shown to mediate the assembly of β-barrel proteins into the OM (15, 23, 27). Accordingly, bacterial OMPs are still recognized by the eukaryotic assembly machinery: when expressed in yeast, bacterial OMPs were found to be assembled into the mitochondrial OM in a Tob55-dependent manner (43). Omp85 in Escherichia coli, which was recently renamed BamA, for β-barrel assembly machinery (Bam) component A, is associated with at least four lipoproteins: BamB (formerly known as YfgL), BamC (NlpB), BamD (YfiO), and BamE (SmpA) (32, 46). In E. coli, BamB, BamC, and BamE are not essential, but the phenotypes of deletion mutants suggest that these proteins contribute to the efficiency of OMP assembly. Like BamA, BamD is an essential protein in E. coli (24, 26), involved in OMP assembly (24). These lipoproteins are evolutionarily less well conserved; the mitochondrial Tob55 protein is associated with two accessory proteins, but they do not show any sequence similarity with the lipoproteins of the E. coli Bam complex (14).Besides E. coli, N. meningitidis is one of the major bacterial model organisms for studies of OM assembly. As mentioned above, it was the first organism in which the function of Omp85 was identified (41), and also, the role of an integral OMP, designated LptD (formerly Imp or OstA), in the transport of lipopolysaccharide (LPS) to the cell surface was first established in N. meningitidis (3). With regard to OM biogenesis, N. meningitidis has several features that distinguish it from E. coli. For example, in contrast to E. coli (13), N. meningitidis mutants defective in LPS synthesis or transport are viable (3, 34), and OMPs are assembled perfectly well in such mutants (33). Furthermore, in OMP assembly mutants of E. coli, the periplasmic accumulation of unassembled OMPs is limited due to the induction of the σ^E extracytoplasmic stress response, which results in the degradation of unfolded OMPs (30) and the inhibition of their synthesis by small regulatory RNAs (20). In contrast, in N. meningitidis, most of the components involved in this response are absent (4), and unassembled OMPs continue to accumulate as periplasmic aggregates when OMP assembly is halted (41). However, the composition of the Bam complex and the role of accessory components in OMP assembly have not so far been studied in this organism. Therefore, to further understand the OMP assembly process in N. meningitidis, we have now analyzed the composition of the Bam complex and addressed the roles of the different components.     

Interplay of protein primary sequence,lipid membrane,and chaperone in β‐barrel assembly

The outer membrane of a Gram‐negative bacterium is a crucial barrier between the external environment and its internal physiology. This barrier is bridged selectively by β‐barrel outer membrane proteins (OMPs). The in vivo folding and biogenesis of OMPs necessitates the assistance of the outer membrane chaperone BamA. Nevertheless, OMPs retain the ability of independent self‐assembly in vitro. Hence, it is unclear whether substrate–chaperone dynamics is influenced by the intrinsic ability of OMPs to fold, the magnitude of BamA–OMP interdependence, and the contribution of BamA to the kinetics of OMP assembly. We addressed this by monitoring the assembly kinetics of multiple 8‐stranded β‐barrel OMP substrates with(out) BamA. We also examined whether BamA is species‐specific, or nonspecifically accelerates folding kinetics of substrates from independent species. Our findings reveal BamA as a substrate‐independent promiscuous molecular chaperone, which assists the unfolded OMP to overcome the kinetic barrier imposed by the bilayer membrane. We additionally show that while BamA kinetically accelerates OMP folding, the OMP primary sequence remains a vital deciding element in its assembly rate. Our study provides unexpected insights on OMP assembly and the functional relevance of BamA in vivo.     

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.     

Involvement and necessity of the Cpx regulon in the event of aberrant β‐barrel outer membrane protein assembly

The Cpx and σ^E regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σ^E pathway monitors the biogenesis of β‐barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β‐barrel OMP mis‐assembly, by utilizing mutants expressing either a defective β‐barrel OMP assembly machinery (Bam) or assembly defective β‐barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σ^E pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β‐barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β‐barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly‐defective β‐barrel OMP species. Together, these results showed that both the Cpx and σ^E regulons are required to reduce envelope stress caused by aberrant β‐barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.     

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.     

The peripheral membrane subunits of the SAM complex function codependently in mitochondrial outer membrane biogenesis

The sorting and assembly machinery (SAM) complex functions in the assembly of beta-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in beta-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that 13 distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required for the SAM complex to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates.