Search

The aim of the present work was to investigate the dynamics of microbial community at DNA and RNA level and the role of bamA and mcrA gene during anaerobic digestion of phenol and p-cresol. Anaerobic digestion was conducted in batch reactors and microbial community dynamics was analysed. Results showed that active microbial community was quite dissimilar in comparison to the total microbial community. Syntrophorhabdus and Bacillus were the dominant active bacterial genera whereas Methanosaeta together with Methanobacterium showed the highest potential activity in the Archaea domain indicating a relevant role of these microorganisms in the anaerobic process. Ecological Networks revealed dissimilar interactions at DNA and RNA level, being the latter a better descriptor of the known roles of dominant OTUs. QRT-PCR results showed that expression of bamA gene correlated positively with instantaneous degradation rate proving for first time its functionality and its relationship with the kinetics of the process.

The β-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae. The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface–displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

Summary The β-barrel assembly machine (BAM) mediates the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria. BamA, the central BAM subunit composed of a transmembrane β-barrel domain linked to five polypeptide transport-associated (POTRA) periplasmic domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. One model is that conformational flexibility between POTRA domains is part of this conformational cycling. Nuclear magnetic resonance (NMR) spectroscopy was used here to study the flexibility of the POTRA domains 1–5 in solution. NMR relaxation studies defined effective rotational correlational times and together with residual dipolar coupling data showed that POTRA1–2 is flexibly linked to POTRA3–5. Mutants of BamA that restrict flexibility between POTRA2 and POTRA3 by disulfide crosslinking displayed impaired function in vivo. Together these data strongly support a model in which conformational cycling of hinge motions between POTRA2 and POTRA3 in BamA is required for biological function.

Significance The outer membrane of Gram-negative bacteria presents a formidable barrier to the discovery of new antibiotics needed to combat infections by multidrug-resistant bacteria. Targeting essential proteins or processes directly exposed to the environment could bypass this obstacle. Here, we describe a monoclonal antibody that selectively and potently antagonizes BamA, which folds and inserts integral outer membrane β-barrel proteins, by binding to a surface-exposed BamA epitope and, as a result, inhibits bacterial cell growth. Mechanisms of resistance to the antibody reveal that membrane fluidity affects BamA activity. This antibody validates the potential therapeutic strategy of targeting essential, exposed functions and provides a powerful tool for dissecting the fundamental process of folding integral membrane β-barrel proteins in vivo., The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Pig has been proven to be a valuable large animal model used for research on diabetic disease. However, their translational value is limited given their distinct anatomy and physiology. For the last 30 years, we have been developing a laboratory Asian miniature pig inbred line (Bama miniature pig, BM) from the primitive Bama xiang pig (BX) via long-term selective inbreeding. Here, we assembled a BM reference genome at full chromosome-scale resolution with a total length of 2.49 Gb. Comparative and evolutionary genomic analyses identified numerous variations between the BM and commercial pig (Duroc), particularly those in the genetic loci associated with the features advantageous to diabetes studies. Resequencing analyses revealed many differentiated gene loci associated with inbreeding and other selective forces. These together with transcriptome analyses of diabetic pig models provide a comprehensive genetic basis for resistance to diabetogenic environment, especially related to energy metabolism.

Summary The β-barrel assembly machine (BAM) integrates β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own β-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of β-barrel proteins, BamA functions as a polypeptide export channel. In vivo structural mapping via intermolecular disulfide crosslinking showed that the extracellular “passenger” domain of a member of the “autotransporter” superfamily of virulence factors traverses the OM through the BamA β-barrel lumen. Furthermore, we demonstrate that a highly conserved residue within autotransporter β-barrels is required to position the passenger inside BamA to initiate translocation and that during translocation, the passenger stabilizes the hybrid-barrel state. Our results not only establish a new function for BamA but also unify the divergent functions of BamA and other “Omp85” superfamily transporters.

Made available in DSpace on 2022-04-29T08:30:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Acinetobacter baumannii is an important nosocomial pathogen. BamA is a protein that belongs to a complex responsible for organizing the proteins on the bacterial outer membrane. In this work, we aimed to evaluate murine immune responses to BamA recombinant protein (rAbBamA) from A. baumannii in an animal model of infection, and to assess cross-reactivity of this target for the development of anti-A. baumannii vaccines or diagnostics. Immunization of mice with rAbBamA elicited high antibody titers and antibody recognition of native A. baumannii BamA. Immunofluorescence also detected binding to the bacterial surface. After challenge, immunized mice demonstrated a 40% survival increase and better bacterial clearance in kidneys. Immunoblot of anti-rAbBamA against other medically relevant bacteria showed binding to proteins of approximately 35 kDa in Klebsiella pneumoniae and Escherichia coli lysates, primarily identified as OmpA and OmpC, respectively. Altogether, our data show that anti-rAbBamA antibodies provide a protective response against A. baumannii infection in mice. However, the response elicited by immunization with rAbBamA is not completely specific to A. baumannii. Although a broad-spectrum vaccine that protects against various pathogens is an appealing strategy, antibody reactivity against the human microbiota is undesired. In fact, immunization with rAbBamA produced noticeable effects on the gut microbiota. However, the changes elicited were small and non-specific, given that no significant changes in the abundance of Proteobacteria were observed. Overall, rAbBamA is a promising target, but specificity must be considered in the development of immunological tools against A. baumannii. Bio-Manguinhos Oswaldo Cruz Foundation Brazilian Ministry of Health Oswaldo Cruz Institute Oswaldo Cruz Foundation Brazilian Ministry of Health National Institute of Science and Technology of Innovation on Diseases of Neglected Populations Center for Technological Development in Health Oswaldo Cruz Foundation São Paulo State University Institute of Biosciences Coastal Campus of São Vicente São Paulo State University Institute of Biosciences Coastal Campus of São Vicente

Aromatic hydrocarbons are usually metabolized to the central intermediate benzoyl-coenzyme A (CoA) prior to ring cleavage. 6-Oxocyclohex-1-ene-1-carbonyl-CoA hydrolase, a key enzyme involved in the reductive benzene ring cleavage, encoded by the bamA gene can serve as a functional biomarker for detecting anaerobic degradation of aromatic compounds in different environments. In the present study, we examined the diversity the bacterial bamA gene in oil field. We sequenced bamA fragments that were PCR amplified from the microbial biomass of two mesophilic oil producing reservoirs during a period of four months. The results showed the presence and a higher diversity of bamA gene than previous studies, indicating its potential involvement in degrading aromatic hydrocarbons through anaerobic biochemical pathways in oil fields. The bamA gene sequences were affiliated to bacteria that are known to degrade aromatics, including Azoarcus, Georgfuchsia, Sulfuritalea, Peptococcaceae and Syntrophus. Differences of composition and diversity of bamA gene sequences over a period of four months in these oil reservoirs showed that it is necessary to monitor functional genes over a period of time to fully establish their significance to any specific process at any site.

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis.

Many locales featuring therapeutic landscapes have seen a rise in health tourism recent years. This study introduces an actor-network perspective to examine the co-evolution of therapeutic landscapes and health tourism, and its inherent dynamism. We argue that therapeutic landscapes and health tourism are emerging out of an integrated actor-network, and thus are in continuous processes of (re)ordering and co-evolution. We also propose a typology of dynamics for the study of such an actor-network, substantiated with an empirical study of the Bama longevity villages in China, in which four interrelated and cascaded dynamics are closely scrutinized: tourists as part of the therapeutic landscape; tourism's impact on the landscape; the heterogeneous therapeutic perceptions of tourists; and the extension of the therapeutic network by health tourism. This study contributes to the relational thinking of therapeutic landscapes and health tourism, and enriches the understanding of their interlacing dynamics from the vantage point of the tourismscape.

This study focused on the stigmatisation of an emerging wellness tourism destination due to patient travel for tourism. The concept of spatial stigma was adopted to explore how local residents perceive, experience and manage the particular negative effects of wellness tourism. The study investigated Bama Yao Autonomous County, colloquially known as ‘Bama’, in China, to which many tourists with cancer and other chronic diseases travel. The results showed that the influx of wellness tourists brought significant challenges in this area. The residents reported ambivalent experiences of and feelings about wellness tourism in local communities, and disagreed with the vilification of wellness tourists. However, they were concerned about the potential consequences of wellness tourism. To manage and resist spatial stigma, the residents deliberately separated themselves from the places occupied by wellness tourists. The theoretical contributions and managerial implications of the study are discussed.

Zinc (Zn) can accumulate in the body of wild animal and human through bio-magnification effects in the food chain to pose chronic toxicity. Male spermatogenesis was sensitive to excessive Zn and elevated temperature. This study aimed to examine whether or not excessive Zn intake caused testicular toxicity and estimate the interaction between Zn and high temperature (HT) in testes of Bama miniature pigs. Six-month-old pigs were pre-fed with or without additional Zn at 1500 mg/kg diet for 30 d and afterward subjected to HT at 40 °C for 5 h daily for 8 consecutive days. Blood samples were collected on d 31 and d 38 and testes were obtained on d 38 immediately after HT exposure. Our data showed both scrotal surface temperature (T) and body surface T increased after 5-h HT exposure (p 0.05). Pigs fed with additional Zn showed germ cell loss, the decreased testes weight (p 0.01) and the elevated testicular H

The outer membranes (OM) of Gram-negative bacteria contain a host of β-barrel outer membrane proteins (OMPs) which serve many functions for cell survival and virulence. The biogenesis of these OMPs is mediated by the β-barrel assembly machinery (BAM) complex which is composed of five components including the essential core component called BamA that mediates the insertase function within the OM. The crystal structure of BamA has recently been reported from three different species, including a full-length structure from Neisseria gonorrhoeae. Mutagenesis and functional studies identified several conformational changes within BamA that are required for function, providing a significant advancement towards unraveling exactly how BamA and the BAM complex are able to fold and insert new OMPs in the OM.

Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.

Variation in the hypothalamic-pituitary-adrenal (HPA) axis has been reported in numerous species and has been implicated in the differences of adrenal response to ACTH observed between different pig breeds; however, few studies have been conducted in indigenous Chinese pig breeds. The present study, we have investigated ACTH regulated adrenal glucocorticoid synthesis pathway in five breeds of pig: four indigenous Chinese pig breeds (Bama, Huanjiang, Lantang and Ningxiang) and an alien breed (Landrace) which were under same conditions. As a result, the ACTH concentrations were significantly greater than others in Lantang and significantly lesser than others in Bama, and they were also significantly lesser in Ningxiang than in Landrace. On the contrary, the cortisol concentrations in Lantang were significantly lesser than those in Bama. According to the opposite pattern on serum concentrations of ACTH and cortisol, Bama, Lantang and Landrace were chosen for further research. The protein contents of MC2R and StAR in adrenal were significantly lesser in Lantang than in Landrace, and the MC2R levels of Bama were also significantly greater than those of Lantang. The StAR protein levels in Bama were greater than those in Lantang as well, and this difference approached significance. Compared with Landrace, the number of mRNA transcripts for StAR, POMC and PC1 in Lantang were significantly lesser than in Landrace. These results indicate Lantang exhibited lower cortisol synthesis capacity under a relatively greater serum ACTH concentration, while Bama and Landrace exhibited just the opposite. These results at least reveals ACTH regulated glucocorticoid synthesis pathway in adrenal of different breeds of pigs, which can provided reference data for further breeding work.

SummaryThe outer membrane of Gram-negative bacteria is replete with a host of β-barrel outer membrane proteins (OMPs). Despite serving a variety of essential functions, including immune response evasion, the exact mechanism of OMP folding and membrane insertion remains largely unclear. The β-barrel assembly machinery complex is required for OMP biogenesis. Crystal structures and molecular dynamics (MD) simulations of the central and essential component, BamA, suggest a mechanism involving lateral opening of its barrel domain. MD simulations reported here reveal an additional feature of BamA: a substrate exit pore positioned above the lateral opening site. Disulfide crosslinks that prevent lateral opening and exit pore formation result in a loss of BamA function, which can be fully rescued by the reductant tris(2-carboxyethyl)phosphine. These data provide strong evidence that lateral opening and exit pore formation are required for BamA function.

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein–protein and protein–lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane. © 2014 Elsevier Ltd. All rights reserved.

Folding and insertion of integral β-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the β-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.

β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex.

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.

Summary Folding and insertion of β-barrel outer membrane proteins (OMPs) is essential for Gram-negative bacteria. This process is mediated by the multiprotein complex BAM, composed of the essential β-barrel OMP BamA and four lipoproteins (BamBCDE). The periplasmic domain of BamA is key for its function and contains five "polypeptide transport-associated" (POTRA) repeats. Here, we report the crystal structure of the POTRA4-5 tandem, containing the essential for BAM complex formation and cell viability POTRA5. The domain orientation observed in the crystal is validated by solution NMR and SAXS. Using previously determined structures of BamA POTRA1-4, we present a spliced model of the entire BamA periplasmic domain validated by SAXS. Solution scattering shows that conformational flexibility between POTRA2 and 3 gives rise to compact and extended conformations. The length of BamA in its extended conformation suggests that the protein may bridge the inner and outer membranes across the periplasmic space.

Insects are among the most successful animals on Earth both with regard to their biomass and biodiversity. It is estimated that up to 20% of all insects are obligately associated with symbiotic microorganisms, and it is likely that their capacity to engage microbial companions has greatly contributed to their evolutionary success. The main focus of this review lies on obligately intracellular bacteria residing in specialized cells, the bacteriocytes, provided by the host. In the past few years the focus in research on these bacteria has been on their biological role for the host and the consequences on the genome and metabolic capacities shaped by a long-lasting obligate association confined to the interior of a eukaryotic host cell. Here, we compare those endosymbiont-host interactions where the genome of the bacterium is sequenced.

The outer membrane of Gram-negative bacteria is an essential asymmetric lipid bilayer that serves as a protective barrier and performs required cellular functions, such as nutrient uptake and export. Within the outer membrane are trans-membrane proteins that adopt unique beta-barrel structures. These outer membrane proteins (Omps) are folded and inserted by a multi-protein complex embedded in the membrane called the Bam complex, which performs this function without the use of chemical energy (i.e. ATP or proton motive force). The Bam complex is made of five proteins, BamA-E. BamA is a beta-barrel itself and is considered the central component of the complex. Crystal structures and molecular dynamics simulations suggests that BamA has a destabilized barrel seam that can open and close to potentially nucleate new beta-barrels. Locking the barrel seam in vivo with engineered disulfides is lethal, suggesting that this lateral gate is essential for BamA function. We tested the lateral gate hypothesis in a minimal in vitro system with BamA and a substrate Omp, OmpX in synthetic liposomes. In this system, locking the barrel has no affect on the activity of BamA – locked barrels had the same activity as unlocked barrels. This suggests that either the lateral gate is not important for function or we are not capturing the full activity of BamA. Interestingly, disulfide cross-linking experiments suggest that the barrel seam is highly dynamic, since disulfide pairs separated by up to 4 residues are able to form. To further probe the lateral gate hypothesis, we have moved into a fully reconstituted system with the Bam complex in native E. coli lipids. This system will help us differentiate between these two possibilities. Understanding how the Bam complex assists in protein folding will help answer fundamental questions about membrane protein folding.

Gram-negative bacteria possess two cell membranes. The outer membrane is host to almost exclusively β-barrel transmembrane proteins. The β-barrel assembly machinery (BAM) is a five-protein complex responsible for the insertion and assembly of outer membrane β-barrel proteins. The first crystal structures of the transmembrane β-barrel domain of BamA were released in 2013. In these initial crystal structures, the BamA β-barrel possessed a weak interface between its first (1) and last (16) β strands, which led to strand separation within 1 μs in our equilibrium simulations. This strand separation has been proposed to act as a lateral gate for substrate passage into the membrane. In addition, a hydrophobic mismatch near the barrel seam was shown to destabilize the membrane, potentially acting to aid integration of the substrate. Furthermore, full BAM complex crystal structures were recently released, showing accessory proteins rallying around this putative insertion region using the periplasmic domains of BamA as a scaffold. In order to address remaining questions surrounding the role that BamA plays in the insertion and assembly process, we have carried out additional equilibrium simulations of BamA in several membrane bilayers. We also performed a calculation of energetic landscapes associated with lateral gate formation under various conditions. These calculations reveal a lower energetic barrier to strand separation for BamA as compared to FhaC, a member of the same (Omp85) family. They also reveal a significant role played by the C-terminal kink feature in modulating the barrier to strand separation. Finally, equilibrium simulations of NgBamA demonstrate lateral gate opening at 340K in a native lipopolysaccharide bilayer for the first time.

For nearly all membrane proteins, insertion into a membrane is not direct, but rather is assisted by other proteins. At the inner membrane, the key element is a protein-conducting channel, the SecY/Sec61 complex. This channel provides a lateral exit into the bilayer while simultaneously offering a pathway into the periplasm. Now, using molecular dynamics flexible fitting (MDFF), we have determined new structures of inactive and active SecY-ribosome complexes. The latter structure reveals in detail how the nascent chain enters the channel, forming loops both above and below it, with the signal anchor intercalated at the open lateral gate. Although bacterial outer-membrane (OM) proteins must first transit SecY into the periplasm, they are inserted via the BamA complex that resides in the OM. Simulations of recently solved crystal structures of BamA suggest that it also opens laterally to the membrane, which has led to the development of a dynamical model for BamA-assisted insertion that is similar in many respect to SecY-mediated insertion.

Bifidobacterium animalis RH is a probiotic bacterium producing exopolysaccharides, which was isolated from the feces of Bama centenarians in Guangxi, China. The genome consists of one chromosome (1,931,057 bp) with no plasmid. The genomic sequence indicated that this strain includes a gene cluster involved in exopolysaccharides (EPS) biosynthesis. Genome sequencing information provides the basis for analysis of molecular mechanisms responsible for regulation of production of EPS.

To develop a bama miniature pig intimal hyperplasia model in superficial femoral artery. Following 1 month of a 3% cholesterol diet, 4 pigs underwent surgical perfusion with distilled water (n=8). 3 pigs were subjected to sham-operation for control (n=6). After 3 months of the same diet, sonography

Elucidating protein plasticity and protein-lipid interactions is critical for a detailed understanding of the workings of membrane protein machines. We study the 88 kDa membrane-embedded protein BamA, which is the main component of the beta-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in gram-negative bacteria. Using a combination of electron microscopy (EM), limited proteolysis and NMR we characterized the fold and dynamics of different BamA constructs and the role of the lipid bilayer for protein structure.Our NMR studies provide atomic insight into protein dynamics and plasticity of the periplasmic POTRA domains and their interactions with the transmembrane domain. Moreover, we studied the influence of lipid type and the lipid-to-protein ratio on the atomic (solid-state NMR) and nm (EM) scale. Taken together, our results provide first clues into the interplay between protein - protein and protein -lipid interactions that may be critical for BAM-mediated substrate insertion into the outer membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide