YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

Author Correction: Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging.

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.

Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6.

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

Relationship between Chemotherapy-Induced Diarrhea and Intestinal Microbiome Composition.

BACKGROUND AND AIM: Fluoropyrimidines (FPs) are key drugs in many chemotherapy regimens; however, recipients are often prone to diarrhea due to gastrointestinal toxicity. Disruption of the intestinal epithelial barrier function by FPs leads to dysbiosis, which may exacerbate intestinal epithelial cell damage as a secondary effect and trigger diarrhea. However, despite studies on chemotherapy-induced changes in the intestinal microbiome of humans, the relationship between dysbiosis and diarrhea is unclear. In this study, we aimed to investigate the relationship between chemotherapy-induced diarrhea and the intestinal microbiome. METHODS: We conducted a single-center prospective observational study. Twenty-three patients who received chemotherapy, including FPs as first-line chemotherapy for colorectal cancer, were included. Stool samples were collected before the start of chemotherapy and after one cycle of treatment to analyze intestinal microbiome composition and perform PICRUSt predictive metagenomic analysis. RESULTS: Gastrointestinal toxicity was observed in 7 of 23 patients (30.4%), diarrhea was observed in 4 (17.4%), and nausea and anorexia were observed in 3 (13.0%). In 19 patients treated with oral FPs, the α diversity of the microbial community decreased significantly following chemotherapy only in the diarrheal group. At the phylum level, the diarrheal group showed a significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Bacteroidetes with chemotherapy (p = 0.013 and 0.011, respectively). In the same groups, at the genus level, Bifidobacterium abundance was significantly decreased (p = 0.019). In contrast, in the non-diarrheal group, Actinobacteria abundance increased significantly with chemotherapy at the phylum level (p = 0.011). Further, Bifidobacterium, Fusicatenibacter, and Dorea abundance significantly increased at the genus level (p = 0.006, 0.019, and 0.011, respectively). The PICRUSt predictive metagenomic analysis revealed that chemotherapy caused significant differences in membrane transport in KEGG pathway level 2 and in 8 KEGG pathway level 3, including transporters and oxidative phosphorylation in the diarrhea group. CONCLUSION: Organic-acid-producing bacteria seem to be involved in diarrhea associated with chemotherapy, including FPs.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective ß-barrel protein degradation.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Alterations in the gut microbiome in patients with esophageal carcinoma in response to esophagectomy and neoadjuvant treatment.

PURPOSE: Analyzing the gut microbiome is essential for planning treatment strategies to manage esophageal squamous cell carcinoma. This study aimed to characterize the gut microbiome of patients with esophageal squamous cell carcinoma and to identify alterations in its composition during treatment. METHODS: We observed alterations in the gut microbiome in 21 consecutive patients with esophageal squamous cell carcinoma at five different time points, from neoadjuvant treatment to postoperative surgery. Ten healthy individuals were used as a non-cancer control group. Fecal samples were collected and analyzed using 16S ribosomal ribonucleic acid sequencing. RESULTS: Before treatment, participants with esophageal squamous cell carcinoma had different alpha and beta diversity in comparison to healthy controls. The number of Streptococcus, a facultative anaerobic bacterium, was significantly higher, whereas that of Faecalibacterium, an obligate anaerobic bacterium, was significantly lower. Both alpha and beta diversity remained unchanged during neoadjuvant treatment, but the alterations were pronounced after surgery. The increase in the relative abundance of Streptococcus and the decrease in that of Faecalibacterium also tended to be more pronounced after surgery. CONCLUSIONS: The gut microbiome in patients with esophageal squamous cell carcinoma is altered with surgical intervention.

Comparison of the Therapeutic Effects of Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells on Renal Fibrosis.

Mesenchymal stem cells (MSCs) have attracted a great deal of interest as a therapeutic tool for renal fibrosis. Although both adipose-derived and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) suppress renal fibrosis, which of these two has a stronger therapeutic effect remains unclear. This study aimed to compare the antifibrotic effects of ADSCs and BMSCs extracted from adipose tissue and bone marrow derived from the same rats. When cultured in serum-containing medium, ADSCs had a more potent inhibitory effect than BMSCs on renal fibrosis induced by ischemia-reperfusion injury in rats. ADSCs and BMSCs cultured in serum-free medium were equally effective in suppressing renal fibrosis. Mice infused with ADSCs (serum-containing or serum-free cultivation) had a higher death rate from pulmonary embolism than those infused with BMSCs. In vitro, mRNA levels of tissue factor, tumor necrosis factor-α-induced protein 6 and prostaglandin E synthase were higher in ADSCs than in BMSCs, while that of vascular endothelial growth factor was higher in BMSCs than in ADSCs. Although ADSCs had a stronger antifibrotic effect, these findings support the consideration of thromboembolism risk in clinical applications. Our results emphasize the importance of deciding between ADSCs and BMSCs based upon the target disease and culture method.

Erratum: Comparative QTL mapping for male sterility of cultivated strawberry (Fragaria × ananassa Duch.) using different reference genome sequences.

[This corrects the article DOI: 10.1270/jsbbs.20151.].

A snapshot of membrane protein insertion.

The cytoplasm is the main place for protein translation from where nascent proteins are transported to their working areas, including the inside, outside, and membrane of the cell. The majority of newly synthesized membrane proteins is co-translationally inserted into the membrane by the evolutionary conserved Sec translocon. In this issue of EMBO Reports, Kater et al [1] use single-particle cryo-electron microscopy to visualize a high-resolution structure of the E. coli SecYEG translocon:ribosome-nascent chain complex in a lipid environment constituted by nanodiscs. This snapshot represents an early intermediate state in membrane protein insertion and provides important information for understanding the molecular mechanism of membrane protein biogenesis.

Dicopper(II) Complexes of p-Cresol-2,6-Bis(dpa) Amide-Tether Ligands: Large Enhancement of Oxidative DNA Cleavage, Cytotoxicity, and Mechanistic Insight by Intracellular Visualization.

Dicopper complexes of a new p-cresol-2,6-bis(dpa) amide-tether ligand (HL1), [Cu2(µ-OH2)(µ-1,3-OAc)(L1)](ClO4)2 (1) and [Cu2(µ-1,1-OAc)(µ-1,3-OAc)(L1)]X (X = ClO4 (2a), OAc (2b)) were synthesized and structurally characterized. 2b rapidly cleaves supercoiled plasmid DNA by activating H2O2 at neutral pH to a linear DNA and shows remarkable cytotoxicity in comparison with related complexes. As 2b is more cytotoxic than HL1, the dicopper core is kept in the cell. A boron dipyrromethene (Bodipy)-modified complex of the p-cresol-2,6-bis(dpa) amide-tether ligand having a Bodipy pendant (HL2), [Cu2(µ-OAc)2(L2)](OAc) (3), was synthesized to visualize intracellular behavior, suggesting that 2b attacks the nucleolus and mitochondria. A comet assay clearly shows that 2b does not cleave nuclear DNA. The apoptotic cell death is evidenced from flow cytometry.

Gut microbiota composition associated with hepatic fibrosis in non-obese patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Gut microbiota composition is associated with the pathogenesis of non-alcoholic fatty liver disease. However, the association between gut microbiota composition and non-alcoholic fatty liver disease in non-obese patients remains unclear. We compared clinical parameters and gut microbiota profiles of healthy controls and non-obese and obese patients with non-alcoholic fatty liver disease. METHODS: We examined the clinical parameters and gut microbiota profiles by 16S rRNA sequences and short-chain fatty acid levels in fecal samples from 51 non-obese patients with non-alcoholic fatty liver disease (body mass index <25 kg/m2 ) and 51 obese patients with non-alcoholic fatty liver disease (body mass index ≥30 kg/m2 ) who underwent pathological examination and 87 controls at five hospitals in Japan. RESULTS: Although no significant differences between the non-obese and other groups were observed in alpha diversity, a significant difference was found in beta diversity. We observed a significant decrease in serum alanine aminotransferase levels, Eubacterium population, and butyric acid levels in non-obese patients with non-alcoholic fatty liver disease compared with those in obese patients with non-alcoholic fatty liver disease. A significant negative correlation was found between the stage of hepatic fibrosis and Eubacterium abundance in non-obese patients with non-alcoholic fatty liver disease. CONCLUSIONS: The decrease in the abundance of Eubacterium that produces butyric acid may play an important role in the development of non-alcoholic fatty liver disease in non-obese individuals. This study was registered at the University Hospital Medical Information Network clinical trial registration system (UMIN000020917).

Comparative QTL mapping for male sterility of cultivated strawberry (Fragaria × ananassa Duch.) using different reference genome sequences.

Male sterility is one of the reproductive isolation systems in plants and quite useful for F1 seed production. We previously identified three independent quantitative trait loci (QTLs) for male sterility of cultivated strawberry, Here, we identified the specific subgenomes in which these QTLs are located by QTL-seq approach. QTLs qMS4.1, qMS4.2, and qMS4.3 were mapped separately in subgenomes Fvb4-4, Fvb4-3, and Fvb4-1, respectively, in 'Camarosa' genome assembly v. 1.0.a1. Candidate regions of qMS4.1 and qMS4.3 were clearly detected around 12-26 Mb in Fvb4-4 and 12-14 Mb in Fvb4-1, respectively; those of qMS4.2 were fragmented in Fvb4-3, which suggests that some scaffolds were incorrectly assembled in Fvb4-3. qMS4.3 was mapped to chr4X1 of 'Reikou' genome assembly r2.3, and qMS4.1 and qMS4.2 were both mapped to chr4Av, which indicates that differentiation of the subgenomes in which both QTLs are located was insufficient in 'Reikou' r2.3. Although 'Camarosa' genome assembly v. 1.0.a1 is an unphased map, which merges homologous chromosomes into one sequence, 'Reikou' genome assembly r2.3 is a phased map, which separates homologous chromosomes. QTL mapping to different reference genomes clearly showed the specific features of each reference genome, and that using different kinds of reference map could accelerate fine mapping and map-based cloning of certain genes of cultivated strawberry.

Structural basis of Sec-independent membrane protein insertion by YidC.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Microscopic Ophthalmic Surgery Using a Freely Movable Arm Support Robot: Basic Experiment and Clinical Experience.

INTRODUCTION: The disadvantage of conventional armrests is the difficulty to adjust their height and position during surgery. OBJECTIVES: We investigated whether a freely movable armrest (FMA) that follows the surgeon's arm is useful for reducing surgeon fatigue and arm tremors and for improving surgical accuracy. METHODS: In the basic study, a corneal suture was placed in porcine eyes with FMA (FMA group) or without FMA (non-FMA group). The suture was intended to pass through two points. Tremor was quantified using motion-tracking software when the needle was held. Subjective symptoms of arm-related fatigue were scored using a questionnaire. Suturing accuracy was compared between the groups. In the clinical study, the same surgeon performed corneal transplantation, cataract surgery, and glaucoma surgery with or without FMA. Fatigue scores from the questionnaire were then compared. RESULTS: In the basic study, hand tremor at rest was significantly improved with FMA (p < 0.0001). The subjective arm fatigue score was significantly reduced (p = 0.0465), while the subjective tremor score was also significantly improved (p = 0.0453). There were no differences in the accuracy of corneal suturing with or without FMA. In the clinical study, the total and arm-related fatigue scores during corneal transplantation and glaucoma surgery tended to improve. CONCLUSIONS: FMA is a useful tool for fixing the surgeon's arm in any position and may improve the quality of ophthalmic surgery by reducing hand tremor and fatigue.

Alteration of Colonic Mucosal Permeability during Antibiotic-Induced Dysbiosis.

Although dysbiosis is likely to disturb the mucosal barrier system, the mechanism involved has remained unclear. Here, we investigated alterations of colonic mucosal permeability and tight junction (TJ) molecules in mice with antibiotic-induced dysbiosis. Mice were orally administered vancomycin or polymyxin B for 7 days, and then fecal samples were subjected to microbial 16S rRNA analysis. The colonic mucosal permeability was evaluated by chamber assay. The colonic expression of TJ molecules and cytokines was examined by real-time RT-PCR, Western blotting, and immunohistochemistry. Caco2 cells were stimulated with cytokines and their transepithelial electric resistance (TEER) was measured. Vancomycin-treated mice showed significantly lower gut microbiota diversity than controls, and the same tendency was evident in polymyxin B-treated mice. The colonic mucosal permeability was significantly elevated in both vancomycin- and polymyxin B-treated mice. The expression of claudin 4 in the colonic mucosa was decreased in both vancomycin- and polymyxin B-treated mice. Colonic expression of TNF-α and/or IFN-γ was significantly increased in mice that had been administered antibiotics. TNF-α and IFN-γ stimulation dose-dependently decreased TEER in Caco2 cells. Antibiotic-induced dysbiosis is correlated with the enhancement in colonic tissue permeability, accompanied by a reduction in claudin 4 expression and enhancement in TNF-α and/or IFN-γ expression in mice.

Probiotic Bifidobacterium bifidum G9-1 ameliorates phytohemagglutinin-induced diarrhea caused by intestinal dysbiosis.

Diarrhea is largely caused by dysbiosis accompanying the hyperproliferation of Escherichia coli (E. coli). While current treatments can resolve the symptoms, they cannot suppress the proliferation of pathogenic bacteria in the intestine. Probiotics have numerous beneficial effects on host health, including restoring the balance of the intestinal microbiota. This study investigated the effect of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1), which is active in intestinal dysbiosis, in the incidence of diarrhea, in the composition of the intestinal microbiota, and in the intestinal tissue of a rat model of phytohemagglutinin (PHA)-induced diarrhea. The rats were treated with PHA, with and without BBG9-1, and the microbiota composition throughout the intestine and stool was examined using high-throughput 16S rRNA sequencing. In line with previous reports, PHA administration caused diarrhea as well as dysbiosis due to E. coli hyperproliferation. Histological findings indicated that the jejunal villus length was shortened. Rats that received BBG9-1 showed clear improvements in dysbiosis, diarrhea symptoms, and jejunal villus length. Principal coordinates analysis demonstrated the microbiota profile to be more similar between the BBG9-1 and normal groups than between the PHA and normal groups. These results indicated that BBG9-1 suppresses the hyperproliferation of E. coli and restores the jejunal villus length, thereby improving dysbiosis, and in turn, alleviating the symptoms of diarrhea.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H(+) or Na(+) across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H(+)-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1-3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

The TPR domain of BepA is required for productive interaction with substrate proteins and the ß-barrel assembly machinery complex.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the ß-barrel assembly machinery (BAM) driving integration of ß-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.