Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli.

Bacterial lipoproteins perform a diverse array of functions including bacterial envelope biogenesis and microbe-host interactions. Lipoproteins in gram-negative bacteria are sorted to the outer membrane (OM) via the localization of lipoproteins (Lol) export pathway. The ATP-binding cassette (ABC) transporter LolCDE initiates the Lol pathway by selectively extracting and transporting lipoproteins for trafficking. Here, we report cryo-EM structures of LolCDE in apo, lipoprotein-bound, and AMPPNP-bound states at a resolution of 3.5 to 4.2 Å. Structure-based disulfide crosslinking, photo-crosslinking, and functional complementation assay verify the apo-state structure and reveal the molecular details regarding substrate selectivity and substrate entry route. Our studies snapshot 3 functional states of LolCDE in a transport cycle, providing deep insights into the mechanisms that underlie LolCDE-mediated lipoprotein sorting in E. coli.

Structures of the ß-barrel assembly machine recognizing outer membrane protein substrates.

ß-barrel outer membrane proteins (ß-OMPs) play critical roles in nutrition acquisition, protein import/export, and other fundamental biological processes. The assembly of ß-OMPs in Gram-negative bacteria is mediated by the ß-barrel assembly machinery (BAM) complex, yet its precise mechanism remains elusive. Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex with bound substrates. Structural analysis indicates that the membrane compositions surrounding the BAM complex could modulate its overall conformations, indicating low energy barriers between different conformational states and a highly dynamic nature of the BAM complex. Importantly, structures of the BAM complex with bound substrates and the related functional analysis show that the first ß-strand of the BamA ß-barrel (ß1BamA ) in the BAM complex is associated with the last but not the first ß-strand of a ß-OMP substrate via antiparallel ß-strand interactions. These observations are consistent with the ß-signal hypothesis during ß-OMP biogenesis, and suggest that the ß1BamA strand in the BAM complex may interact with the last ß-strand of an incoming ß-OMP substrate upon their release from the chaperone-bound state.

Bacillus fonticola sp. nov., isolated from deep sea cold seep sediment.

In this study, we report a novel Gram-positive bacterium, designated as strain CS13T, isolated from deep-sea sediment collected in the cold seep area of the South China Sea. Growth of strain CS13T occurred at 16-37 °C (optimum 25-28 °C), pH 7.0-9.0 (optimum, 7.0), and 0-8% (w/v) NaCl (optimum, 2-3%). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain CS13T belonged to the genus Bacillus. The closest phylogenetic neighbors of strain CS13T are Bacillus carboniphilus JCM 9731T (96.0%), Bacillus pakistanensis NCCP-168T (95.7%) and Bacillus acidicola 105-2T (95.6%). The genomic DNA G + C content of strain CS13T is 43.7 mol%. The principal respiratory quinone was menaquinone 7 (MK-7). The polar lipids of CS13T contained diphosphatidylglycerol, phosphatidylglycerol, phospholipid, and glycolipid. The major fatty acids of CS13T contained anteiso-C15:0, anteiso-C17:0, C16:0 and C18:0. Strain CS13T harboured meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic, physiological, biochemical, and morphological analyses suggested that strain CS13T represents a novel species of genus Bacillus, and the name Bacillus fonticola sp. nov. is proposed for the type species CS13T (= CCTCC AB 2019194T = JCM 33663T).

Description of Novosphingopyxis iocasae sp. nov., isolated from deep sea sediment from the Mariana Trench, and emended description of the genus Novosphingopyxis.

In this study, we reported a Gram-stain-negative, orange-coloured, rod-shaped, motile and faculatively anaerobic bacterium named strain PB63T, which was isolated from the deep-sea sediment from the Mariana Trench. Growth of PB63T occurred at 10-35 °C (optimum, 28 °C), pH 5.0-8.0 (optimum, 5.0-6.0) and with 0-7 % (w/v) NaCl (optimum, 2-3 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that PB63T represented a member of the genus Novosphingopyxis and was closely related to Novosphingopyxis baekryungensis DSM 16222T (97.9 % sequence similarity). PB63T showed tolerance to a variety of heavy metals, including Co2+, Zn2+, Mn2+ and Cu2+. The complete genome of PB63T was obtained, and many genes involved in heavy metal resistance were found. The genomic DNA G+C content of PB63T was 62.8 mol%. The predominant respiratory quinone of PB63T was ubiquinone-10 (Q-10). The polar lipids of PB63T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, glycolipid, phosphatidylcholines and three unidentified lipids. The major fatty acids of PB63T included summed feature 8 (C18 : 1ω7c or/and C18 : 1ω6c), C14 : 0 2-OH, 11-methyl C18 : 1ω7c, C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C17 : 1ω6c. The results of phylogenetic, physiological, biochemical and morphological analyses indicated that strain PB63T represents a novel species of the genus Novosphingopyxis, and the name Novosphingopyxis iocasae sp. nov. is proposed with the type species PB63T (=CCTCC AB 2019195T=JCM 34178T).

Outbreak of COVID-19 in a family, Wenzhou, China.

Since December 2019, China has experienced a widespread outbreak of COVID-19. However, at the early stage of outbreak, investigations revealed a variety of patterns resulting in the transmission of COVID-19. Thus, it is essential to understand the transmission types and the potential for sustained human-to-human transmission. Moreover, the information regarding the characteristics of transmission helps in coordinating the current screening programme, and controlling and containing measures, and also, helps in deciding the appropriate quarantine duration. Thus, this investigation reports an outbreak of COVID-19 in a family residing in Wenzhou, Zhejiang, China during the month of January-February 2020.

Conformational Dynamics, Intramolecular Domain Conformation Signaling, and Activation of Apo-FimD Revealed by Single-Molecule Fluorescence Resonance Energy Transfer Studies.

The chaperone-usher secretion pathway is a conserved bacterial protein secretion system dedicated to the biogenesis of adhesive fibers. Usher, a multidomain-containing outer membrane protein, plays a central role in this process by acting as a molecular machine that recruits different chaperone-subunit complexes, catalyzes subunit polymerization, and forms a channel for secretion of the assembled subunits. While recent crystal structural studies have greatly advanced our understanding of the structure and function of ushers, the overall architecture of the full-length apo-usher, the molecular events that dictate conformational changes in usher during pilus biogenesis, and its activation by the specific chaperone-adhesin complex remain largely elusive. Using single-molecule fluorescence resonance energy transfer studies, we found that the substrate-free usher FimD (apo-FimD) adopts a contracted conformation that is distinct from its substrate-bound states; both the N-terminal domain (NTD) and the C-terminal domain (CTD) of apo-FimD are highly dynamic, and FimD coordinates its domain conformational changes via intramolecular domain conformation signaling. By combining these studies with in vitro photo-cross-linking studies, we further show that only the chaperone-bound adhesin (FimC:FimH) can be transferred to the CTD, dislocates the plug domain, and triggers conformational changes in the remaining FimD domains. Taken together, these studies delineate an overall architecture of the full-length apo-FimD, provide detailed mechanic insight into the activation of apo-FimD, and explain why FimD could adjust its conformational states to perform multiple functions in each cycle of pilus subunit addition and ensure that pilus assembly proceeds progressively in a cellular energy-free environment.

Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins represents a unique membrane protein family that facilitates the insertion, folding, and assembly of a cohort of α-helical membrane proteins in all kingdoms of life, yet its underlying mechanisms remain elusive. We report the crystal structures of the full-length Thermotoga maritima YidC (TmYidC) and the TmYidC periplasmic domain (TmPD) at a resolution of 3.8 and 2.5 Å, respectively. The crystal structure of TmPD reveals a ß-supersandwich fold but with apparently shortened ß strands and different connectivity, as compared to the Escherichia coli YidC (EcYidC) periplasmic domain (EcPD). TmYidC in a detergent-solubilized state also adopts a monomeric form and its conserved core domain, which consists of 2 loosely associated α-helical bundles, assemble a fold similar to that of the other YidC homologues, yet distinct from that of the archaeal YidC-like DUF106 protein. Functional analysis using in vivo photo-crosslinking experiments demonstrates that Pf3 coat protein, a Sec-independent YidC substrate, exits to the lipid bilayer laterally via one of the 2 α-helical bundle interfaces: TM3-TM5. Engineered intramolecular disulfide bonds in TmYidC, in combination with complementation assays, suggest that significant rearrangement of the 2 α-helical bundles at the top of the hydrophilic groove is critical for TmYidC function. These experiments provide a more detailed mechanical insight into YidC-mediated membrane protein biogenesis.-Xin, Y., Zhao, Y., Zheng, J., Zhou, H., Zhang, X. C., Tian, C., Huang, Y. Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

Global discovery of small RNAs in the fish pathogen Edwardsiella piscicida: key regulator of adversity and pathogenicity.

Recently, bacterial small RNA (sRNA) has been shown to be involved as a key regulator in stress responses. sRNAs of Edwardsiella piscicida, an important aquatic pathogen, are not well characterized to date. In this study, using RNA-seq technology, we globally found and identified sRNA candidates expressed from E. piscicida grown in normal LB medium, acid pressure, iron deficiency stress, and oxidation pressure. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA candidates by searching against the Rfam database. Compared in normal condition, the expression of 103 sRNAs (DEsRNA, differentially expressed sRNA) and 1615 mRNAs (DEmRNAs, differentially expressed mRNA) showed significant differences in three stress sample. Based on the prediction by IntaRNA and relational analysis between DEsRNAs and DEmRNAs, 103 DEsRNAs were predicted to regulate 769 target mRNAs. Pleiotropic function of target DEmRNAs indicated that sRNAs extensively participated in a variety of physiological processes, including response to adversity and pathogenicity, the latter was further confirmed by infection experiment. A large number transcription factors appeared in target genes of sRNAs, which suggested that sRNAs likely deeply interlaced within complex gene regulatory networks of E. piscicida. Moreover, 49 Hfq-associated sRNAs were also identified in this study. In summary, we globally discovered sRNAs for the first time in pathogenic bacteria of fish, and our findings indicated that sRNAs in E. piscicida have important roles in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane.

The partially de-N-acetylated poly-ß-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane ß-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the ß-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane.

MicroRNA-495 Inhibits New Bone Regeneration via Targeting High Mobility Group AT-Hook 2 (HMGA2).

BACKGROUND MicroRNAs play critical roles in post-translational gene expression. In this study, we explored the role of miR-495 in new bone regeneration. MATERIAL AND METHODS Murine calvarial osteoblasts were isolated and cultured. Microarray was performed to identify differential miRNAs in medicarpin-induced osteoblasts differentiation. Luciferase reporter assay was performed to identify the target gene of miRNA. Murine osteoblast cells were transfected with miC, miR-495, or anti-miR-495. CCK-8 and flow cytometry were performed to detect osteoblasts proliferation and apoptosis. Western blot was used to analyze apoptosis-related proteins. qRT-PCR analysis was performed to detect gene expression. ALP activity and mineralized nodule formation test were used to evaluate bone formation. Dill-hole injury model was constructed and micro CT was utilized to measuring bone healing. RESULTS Microarray analysis identified miR-495 as our miRNA of interest and luciferase reporter assay identified HMGA2 as its target gene. Over-expression of miR-495 significantly inhibited ALP activity and mineralized nodule formation as well as the expression of RUNX-2, BMP-2, and Osterix. Also, miR-495 over-expression inhibited osteoblasts proliferation and promoted apoptosis obviously. In this in vivo study, the downregulation of miR-495 promoted murine femur healing. CONCLUSIONS MiR-495 inhibits new bone regeneration via targeting high mobility group AT-Hook 2 (HMGA2). We propose that targeting miR-495 may be a promising therapeutic approach for bone regeneration.

Structural and functional analysis of the ß-barrel domain of BamA from Escherichia coli.

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a ß-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 ß barrel is composed of 5 markedly short ß strands, and the loops stemming from these ß 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 ß barrel and structure-based mutagenesis studies suggest that the transmembrane ß strands of OMP substrates may integrate into the outer membrane at the interface of the first and last ß 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.

Surgical treatment of scoliosis associated with syringomyelia with no or minor neurologic symptom.

PURPOSE: Syringomyelia with coexisting intraspinal abnormities is thought to increase the risk of neurologic injury during surgical correction of the scoliosis. However, surgical treatment for intraspinal abnormities carries significant morbidity risks including worsening neurological function and wound complications. The authors' aim in this study was to evaluate one-stage posterior correction of scoliosis in this patient population without prophylactic surgical treatment for neural axis malformations before scoliosis correction. METHODS: A total of 29 patients with syringomyelia and coexisting intraspinal abnormities who underwent scoliosis correction were evaluated. The average age was 15.6 years (range 12-23). All patients were examined for neural axis abnormalities using MRI, including syringomyelia with Chiari I malformation in 18 patients, syringomyelia with tethered cord and/or diastematomyelia in 11. None of patients presented with symptoms suggesting significant neurological dysfunction. The surgical efficacies and complications of correction were reviewed. RESULTS: The preoperative Cobb angle of major coronal curve averaged 65° (range 46°-95°), and it measured 28° (range 22°-43°) at the last follow-up, for a 63 % correction. Maximal kyphosis averaged 52° (range 41°-69°) preoperatively, and improved to 29° (range 22°-43°) at ultimate follow-up, for a 46 % correction. The average follow-up was 6 years (4-8 years). None of the patients experienced deterioration in their neurologic status. CONCLUSION: The study results suggested that prophylactic neurosurgery for intraspinal abnormality may be unnecessary in patients with asymptomatic or minor symptomatic syringomyelia and coexisting intraspinal abnormities. One-stage posterior correction of scoliosis in this patient population does not involve significant complications and seems to be an alternative and safe treatment option.

Clinical analysis of 73 cases of intraspinal nerve sheath tumor.

Seventy-three patients with spinal nerve sheath tumor who were surgically treated in our hospital during the years 2004-2010 were retrospectively reviewed with respect to recovery of neurological function, recurrence of the tumor and occurrence of kyphotic deformities. Preoperative clinical manifestations, imaging data, surgical records and follow-up results were comprehensively analyzed. The follow-up duration was 12-60 months with an average of 32.0 months. Out of the 73 cases enrolled, 69 had gradual recovery of sensation, motor and sphincter functions 1 week to 3 months after operation. Forty-six cases had incomplete paralysis, whose American Spinal Injury Association (ASIA) grades, however, were gradually increased during the follow-up period, 4 cases had no significant improvement of the clinical symptoms and no change in ASIA grades during the follow-up period. Two cases had postoperative recurrence of the tumor. There were no deaths, no spinal instability, and no kyphotic malformations found in any cases. Our study indicated that complete removal of the tumor is important for good recovery, and an ideal surgical method may reduce the recurrence of the tumor or the occurrence of complications.

Analgesic comparison between perineural and intravenous dexamethasone for shoulder arthroscopy: a meta-analysis of randomized controlled trials.

INTRODUCTION: The analgesic comparison between perineural and intravenous dexamethasone on interscalene block for pain management after shoulder arthroscopy remains controversial. We conduct this meta-analysis to explore the influence of perineural versus intravenous dexamethasone on interscalene block for pain control after shoulder arthroscopy. METHODS: We have searched PubMed, Embase, Web of science, EBSCO and Cochrane library databases through April 2021 and included randomized controlled trials (RCTs) assessing the effect of perineural and intravenous dexamethasone on interscalene block in patients with shoulder arthroscopy. RESULTS: Five RCTs were included in the meta-analysis. Overall, compared with intravenous dexamethasone for shoulder arthroscopy, perineural dexamethasone led to similar block duration (SMD = 0.12; 95% CI - 0.12 to 0.35; P = 0.33), pain scores at 12 h (SMD = - 0.67; 95% CI - 1.48 to 0.15; P = 0.11), pain scores at 24 h (SMD = - 0.33; 95% CI - 0.79 to 0.14; P = 0.17), opioid consumption (SMD = 0.01; 95% CI - 0.18 to 0.19; P = 0.95) and incidence of nausea/vomiting (OR = 0.74; 95% CI 0.38-1.44; P = 0.38). CONCLUSIONS: Perineural and intravenous dexamethasone demonstrated comparable pain relief after shoulder arthroscopy.

Efficacy of ifosfamide combined with liposome doxorubicin on osteosarcoma and its effects on serum IL-10, TNF-α, and IFN-γ in patients with osteosarcoma.

OBJECTIVE: To evaluate the effects of ifosfamide combined with liposome doxorubicin on osteosarcoma (OS) and its effects on serum IL-10, TNF-α, and IFN-γ in patients with OS. METHODS: A total of 86 patients with OS who received chemotherapy in Honghui Hospital, Xi'an Jiaotong University from Jan. 2017 to Dec. 2019 were enrolled. Patients treated by conventional doxorubicin + ifosfamide were assigned to the regular group (n=40). Others treated by liposome doxorubicin + ifosfamide were assigned to the research group (n=46). The clinical efficacy, 2-year survival rate, and adverse reactions of the two groups were evaluated and compared. ELISA was adopted for quantification of tumor specific growth factor (TSGF), vascular endothelial growth factor (VEGF), erb-b2 receptor tyrosine kinase 3 (ERBB3), tumor necrosis factor-α (TNF-α), interferon-gamma-γ (IFN-γ), and interleukin-10 (IL-10). The EORTC Quality of Life Questionnaire (QLQ-C30) was adopted to evaluate a patient's life quality. RESULTS: The research group showed a higher total effective rate and a higher 2-year survival rate than the regular group, but lower incidences of liver and kidney function injury, thrombocytopenia, and cardiotoxicity than the regular group. After therapy, lower levels of serum TSGF, VEGF, ERBB3, and TNF-α were found in the research group than those in the regular group. Higher levels of IFN-γ and IL-10 were found in the former than those in the latter. The research group got higher scores of QLQ-C30 than the regular group. CONCLUSION: Liposome doxorubicin + ifosfamide can improve the clinical efficacy on patients with OS and improve their recovery and life quality.

MicroRNA-545-5p regulates apoptosis, migration and invasion of osteosarcoma by targeting dimethyladenosine transferase 1.

The metastasis of osteosarcoma is a major threat to both adolescents and young adults. Identifying novel targets that may prevent osteosarcoma metastasis is critical in developing advanced clinical therapies for treating this cancer. The present study aimed to explore the mechanism of microRNA (miR)-545-5p in the metastasis of osteosarcoma. The present study identified miR-545-5p as a potential target that was downregulated in both osteosarcoma clinical samples and cell lines, and in the latter, ectopically expressed miR-545-5p caused apoptosis. In addition, miR-545-5p exerted inhibitory effects in osteosarcoma migration and invasion. Overexpression of miR-545-5p induced xenograft growth inhibition in vivo. In addition, miR-545-5p targeted dimethyladenosine transferase 1 (DIMT1), an oncogenic protein that facilitates osteosarcoma proliferation, migration and invasion. Taken together, the results of the present study suggest that miR-545-5p functions as a tumor suppressor in osteosarcoma that promotes apoptosis, while inhibiting migration and invasion by targeting DIMT1. Taken together, the results of the present study suggest two potential novel targets for osteosarcoma treatment and metastasis prevention.

Multiscale Modification of Populus cathayana by Alkali Lignin Combined with Heat Treatment.

Chemical modification of wood with green modifiers is highly desirable for sustainable development. With the aim of enhancing the water resistance and dimensional stability of fast growing wood, modifications were conducted using renewable and toxicity-free industrial lignin combined with heat treatment. Poplar (Populus cathayana) samples first underwent impregnation with alkali lignin solution and were then subjected to heat treatment at 140⁻180 °C for two hours. The results indicated that the modified wood showed excellent leaching resistance. The alkali lignin treatment improved surface hydrophobicity and compression strength, and decreased moisture and water uptake, thereby reducing the dimensional instability of modified wood. These changes became more pronounced as the heat-treating temperature increased. Scanning electron microscopy, confocal laser scanning microscopy, and Fourier transform infrared spectroscopy evidenced that a multiscale improvement of the alkali lignin occurred in the cell lumen and cell wall of wood fibers and vessels, with small alkali lignin molecules reacting with the wood matrix. This study paves the way for developing an effective modification approach for fast growing wood, as well as promoting the reuse of industrial lignin for high-value applications, and improves the sustainable development of the forestry industry.

The serine protease autotransporter Tsh contributes to the virulence of Edwardsiella tarda.

The temperature-sensitive hemagglutinin (Tsh), identified as serine protease autotransporters of the Enterobacteriaceae (SPATEs) proteins, is an important virulence factor for avian-pathogenic Escherichia coli (APEC) and uropathogenic E. coli. However, little is known about the role of Tsh as a virulence factor in Edwardsiella tarda, a severe fish pathogen. In this study, we characterized the Tsh of E. tarda (named TshEt) and examined its function and vaccine potential. TshEt is composed of 1224 residues and has three functional domains typical for autotransporters. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tshEt was upregulated under conditions of high temperature, increased cell density, high pH, and iron starvation and during the infection of host cells. A markerless tsh in-frame mutant strain, TX01Δtsh, was constructed to determine whether TshEt participates in the pathogenicity of E. tarda, Compared to the wild type TX01, TX01Δtsh exhibited (i) retarded biofilm growth, (ii) decreased resistance against serum killing, (iii) impaired ability to block the host immune response, (iv) attenuated tissue and cellular infectivity. Introduction of a trans-expressed tsh gene restored the lost virulence of TX01Δtsh. The passenger domain of TshEt contains a putative serine protease (PepS) that exhibits apparent proteolytic activity when expressed in and purified from E. coli as a recombinant protein (rPepS). When used as a subunit vaccine to immunize Japanese flounder, rPepS was able to induce effective immune protection. This is the first study of Tsh in a fish pathogen, and the results suggest that TshEt exerts pleiotropic effects on the pathogenesis of E. tarda.

Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins.

In Gram-negative bacteria, the assembly of ß-barrel outer-membrane proteins (OMPs) requires the ß-barrel-assembly machinery (BAM) complex. We determined the crystal structure of the 200-kDa BAM complex from Escherichia coli at 3.55-Å resolution. The structure revealed that the BAM complex assembles into a hat-like shape, in which the BamA ß-barrel domain forms the hat's crown embedded in the outer membrane, and its five polypeptide transport-associated (POTRA) domains interact with the four lipoproteins BamB, BamC, BamD and BamE, thus forming the hat's brim in the periplasm. The assembly of the BAM complex creates a ring-like apparatus beneath the BamA ß-barrel in the periplasm and a potential substrate-exit pore located at the outer membrane-periplasm interface. The complex structure suggests that the chaperone-bound OMP substrates may feed into the chamber of the ring-like apparatus and insert into the outer membrane via the potential substrate-exit pore in an energy-independent manner.