Characterization of the OMP biogenesis machinery in Fusobacterium nucleatum.

The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of β-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular "passenger" domain is linked to a β-barrel that inserts in the OM. Despite their structural and functional diversity, OMPs interact in the periplasm with a relatively small set of protein chaperones that facilitate their transport from the inner membrane (IM) to the β-barrel assembly machinery (BAM complex), preventing aggregation and assisting their folding in various aspects including disulfide bond formation. This chapter is focused on the periplasmic folding factors involved in the biogenesis of integral OMPs and members of T5SS in E. coli, which are used as a model system in this field. Background information on these periplasmic folding factors is provided along with genetic methods to generate conditional mutants that deplete these factors from E. coli and biochemical methods to analyze the folding, surface display, disulfide formation and oligomerization state of OMPs/T5SS in these mutants.

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.

The outer membrane of Gram-negative bacteria is closely related to the pathogenicity and drug resistance of bacteria. Outer membrane proteins (OMPs) are a class of proteins with important biological functions on the outer membrane. The β-barrel assembly machinery (BAM) complex plays a key role in OMP biogenesis, which ensures that the OMP is inserted into the outer membrane in a correct folding manner and performs nutrient uptake, antibiotic resistance, cell adhesion, cell signaling, and maintenance of membrane stability and other functions. The BAM complex is highly conserved among Gram-negative bacteria. The abnormality of the BAM complex will lead to the obstruction of OMP folding, affect the function of the outer membrane, and eventually lead to bacterial death. In view of the important role of the BAM complex in OMP biogenesis, the BAM complex has become an attractive target for the development of new antibacterial drugs against Gram-negative bacteria. Here, we summarize the structure and function of the BAM complex and review the latest research progress of antibacterial drugs targeting BAM in order to provide a new perspective for the development of antibiotics.

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

Bacterial outer membrane proteins (OMPs) contain a unique “β barrel” segment that is inserted into the membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly and were effectively engaged by fewer periplasmic chaperones than their urea-denatured counterparts. Taken together, our results strongly suggest that the mode of production influences the conformational states sampled by OMPs and thereby affects their recognition by both chaperones and the Bam complex. Besides providing insights into OMP biogenesis, our work describes a novel, streamlined method to reconstitute OMP assembly in vitro.

Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM

A Computational Model for Membrane Protein Flux across the Bacterial Periplasm

Characterization of the β-barrel assembly machinery in fusobacterium.

Proteins of the mitochondrial outer membrane are synthesized as precursors on cytosolic ribosomes and sorted via internal targeting sequences to mitochondria. Two different types of integral outer membrane proteins exist: proteins with a transmembrane β-barrel and proteins embedded by a single or multiple α-helices. The import pathways of these two types of membrane proteins differ fundamentally. Precursors of β-barrel proteins are first imported across the outer membrane via the translocase of the outer membrane (TOM complex). The TOM complex is coupled to the sorting and assembly machinery (SAM complex), which catalyzes folding and membrane insertion of these precursors. The mitochondrial import machinery (MIM complex) promotes import of proteins with multiple α-helical membrane spans. Depending on the topology precursors of proteins with a single α-helical membrane anchor are imported via several distinct routes. We summarize current models and open questions of biogenesis of mitochondrial outer membrane proteins and discuss the impact of malfunctions of protein sorting on the development of diseases.

The β-barrel assembly machinery (Bam) is essential for assembling all transmembrane β-barrel outer membrane proteins in gram-negative bacteria. The Bam complex consists of the central β-barrel protein BamA and accessory Bam lipoproteins, including the widely conserved and essential BamD. BamD is assumed to be an essential regulator of OMP assembly, as its absence causes a global defect in OMP biogenesis. Here, we challenge this view by demonstrating that BamD essentiality is both conditional and substrate specific. In Escherichia coli, its function can be bypassed by preventing BamA jamming by a single, non-essential substrate RcsF. Our findings suggest that BamD plays two distinct roles in the Bam complex. It prevents improper RcsF engagement that can jam BamA, and it kinetically enhances BamA-mediated OMP assembly. Contrary to prevailing models, we demonstrate that the second function in general OMP assembly is not essential. We report a genetic background in which each Bam lipoprotein is dispensable for viability, providing a powerful new system for investigating their functions in OMP assembly in the context of unmodified, wild-type BamA.IMPORTANCEThe β-barrel assembly machinery (Bam) complex assembles all outer membrane proteins (OMPs) and is conserved and essential across all gram-negative bacteria. While BamA is critical for the folding and insertion of OMPs into the outer membrane, BamD is also considered essential for OMP assembly because its loss leads to a global reduction in OMP levels and cell death. Our results show that BamD is important but not essential for general OMP assembly. In Escherichia coli, BamD's essentiality arises from its role in preventing a single challenging substrate from jamming BamA and indirectly abolishing OMP assembly. This work challenges long-standing assumptions about BamD's function, underscores the substrate-specific roles of accessory Bam components, and offers important new considerations for interpreting genetic studies of the Bam complex.

The β-barrel assembly machinery (BAM) complex is essential for the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria, with the membrane protein BamA acting as a catalyst for folding of OMPs in the outer membrane. Recently, structures of the BAM complex have been solved, displaying the molecular organization of the five proteins of the complex (BamABCDE). However, the mechanism by which BamA completes its insertase role is unclear. This PhD thesis focuses on the optimization of sample preparation and backbone assignment of the BamA β-barrel domain for solution NMR spectroscopy. Initial NMR spectra of the BamA β-barrel showed broad peaks with a low signal-to-noise ratio. This was likely due to a dynamic nature of the gate-region, as revealed by cysteine-scanning experiments. Therefore, as a first step to obtain a sample of BamA β-barrel suitable for NMR spectroscopy, buffer conditions were optimized. Then, in order to reduce the dynamics of the BamA β-barrel, a construct was designed with a C-terminal extension by nine residues. In addition to this extension, the G433A mutation in the gate-region was identified as to improve the quality of the NMR spectra. At that point, a combination of specific isotopic labeling and unlabeling in auxotrophic strains, triple-resonance experiments and 3D NOESY experiments allowed to obtain sequence-specific NMR resonance assignments of a large portion of the BamA β-barrel in LDAO micelles. The assignments revealed that some residues of the BamA β-barrel were found in different conformations that can be stabilized by the formation of a disulfide bond or by the C-terminal extension. Moreover, the crystal structure of the extended BamA β-barrel was determined, revealing a longer, and therefore more stable β-strand formed between the first and last strand of the barrel, explaining the stabilizing effect observed in its NMR spectrum. Additional work was performed on the soluble proteins of the BAM complex (BamBCDE). The expression and purification of BamB, BamC BamD and BamE was optimized and NMR spectra were recorded. BamD was found to be unstable once purified, and quickly precipitated, preventing to reach a molar concentration suitable for NMR spectroscopy. As a way to circumvent this issue, a hybrid construct of BamCD was prepared. The protein was able to reach high concentrations while keeping its stability. The fingerprint spectrum of BamCD was recorded and the peaks belonging to BamD overlapped with the peaks measured from a sample of individual BamD. This stabilized sample opens the possibility to obtain the sequence-specific assignments of BamD. Overall, this work resulted in the assignment of a large portion of the BamA β-barrel. As BamA is a potential target for new antibiotics, this assignment opens a way to perform NMR studies on BamA with substrates and ligands and understand the mechanical implications of their binding. Additionally, the dynamic nature of the BamA β-barrel was demonstrated by observing multiple conformations with solution-state NMR spectroscopy. In combination with the available assignment, it will be possible to observe the effects of binding molecules, mutations, or of the molecular environment on the conformational ensemble of BamA.

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.

BamA interacts with the BamBCDE lipoproteins, and together they constitute the essential β-barrel assembly machine (BAM) of Escherichia coli. The simultaneous absence of BamB and BamE confers a conditional lethal phenotype and a severe β-barrel outer membrane protein (OMP) biogenesis defect. Without BamB and BamE, wild-type BamA levels are significantly reduced, and the folding of the BamA β-barrel, as assessed by the heat-modifiability assay, is drastically compromised. Single-amino-acid substitutions in the β-barrel domain of BamA improve both bacterial growth and OMP biogenesis in a bamB bamE mutant and restore BamA levels close to the BamB(+) BamE(+) level. The substitutions alter BamA β-barrel folding, and folding in the mutants becomes independent of BamB and BamE. Remarkably, BamA β-barrel alterations also improve OMP biogenesis in cells lacking the major periplasmic chaperone, SurA, which, together with BamB, is thought to facilitate the transfer of partially folded OMPs to the soluble POTRA (polypeptide-transport-associated) domain of BamA. Unlike the bamB bamE mutant background, the absence of BamB or SurA does not affect BamA β-barrel folding. Thus, substitutions in the outer membrane-embedded BamA β-barrel domain overcome OMP biogenesis defects that occur at the POTRA domain of BamA in the periplasm. Based on the structure of FhaC, the altered BamA residues are predicted to lie on a highly conserved loop that folds inside the β-barrel and in regions pointing outside the β-barrel, suggesting that they influence BamA function by both direct and indirect mechanisms.

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.