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The global public health burden of bacterial antimicrobial resistance (AMR) is intensified by Gram-negative bacteria, which have an additional membrane, the outer membrane (OM), outside of the peptidoglycan (PG) cell wall. Bacterial two-component systems (TCSs) aid in maintaining envelope integrity through a phosphorylation cascade by controlling gene expression through sensor kinases and response regulators. In Escherichia coli, the major TCSs defending cells from envelope stress and adaptation are Rcs and Cpx, which are aided by OM lipoproteins RcsF and NlpE as sensors, respectively. In this review, we focus on these two OM sensors. β-Barrel assembly machinery (BAM) inserts transmembrane OM proteins (OMPs) into the OM. BAM co-assembles RcsF, the Rcs sensor, with OMPs, forming the RcsF-OMP complex. Researchers have presented two models for stress sensing in the Rcs pathway. The first model suggests that LPS perturbation stress disassembles the RcsF-OMP complex, freeing RcsF to activate Rcs. The second model proposes that BAM cannot assemble RcsF into OMPs when the OM or PG is under specific stresses, and thus, the unassembled RcsF activates Rcs. These two models may not be mutually exclusive. Here, we evaluate these two models critically in order to elucidate the stress sensing mechanism. NlpE, the Cpx sensor, has an N-terminal (NTD) and a C-terminal domain (CTD). A defect in lipoprotein trafficking results in NlpE retention in the inner membrane, provoking the Cpx response. Signaling requires the NlpE NTD, but not the NlpE CTD; however, OM-anchored NlpE senses adherence to a hydrophobic surface, with the NlpE CTD playing a key role in this function.

To compare the transcriptional differences between the T2DM-susceptible and T2DM-tolerant Bama mini-pigs and to determine the utility of Bama mini-pigs as an animal model for T2DM by comparing the transcriptomes of the animals with T2DM patients. The skeletal muscle transcriptomes of healthy Guangxi Bama mini-pigs (CON), T2DM pigs (T2D), and non-T2DM pigs (NT2D) were determined by RNA sequencing. A total of 290 differentially expressed genes (DEGs) were detected in NT2D relative to the CON groups, 572 DEGs in T2D compared to CON, and 300 DEGs in T2D compared to NT2D. The RNA-seq data was verified by qPCR analysis of PGC1α, NR4A3, CSRP3, MYH7, MYH2, MYH1, GLUT4, PPARγ, LEP, ATP5H, UCP2, and MTOR transcripts. The DEGs in T2D Bama mini-pigs were mainly involved in signaling pathways associated with metabolism, cardiovascular diseases, infectious disease, cancer, inflammation and immune responses, cytoskeleton, and signal transduction. Comparative analysis of the transcriptomes of T2DM Bama mini-pigs and T2DM patients (GSE29221 dataset) revealed 17 differentially co-expressed genes that were assigned to 11 GO terms and 6 annotated pathways, including hypertrophic cardiomyopathy, dilated cardiomyopathy, thyroid cancer, cardiac muscle contraction, tight junction, and calcium signaling pathway, suggesting that the T2D Bama mini-pigs could be used on T2DM research in those pathways. This study represents the first RNAseq transcriptome analysis in T2D Bama mini-pigs and provided a new perspective for the study on the human T2DM pathogenesis by using the T2DM model of Bama mini-pigs.

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure1,2. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

Poly(vinyl alcohol) (PVA) was modified with 3-aminopropyltriethoxysilane and was used for synthesis of poly(urethane-urea) microcapsules (PUUMc) with encapsulated maltogenic a-amylase (BaMa) (E. C. 3.2.1.133). The optimization of PVA modification factors and PUUMc synthesis conditions was performed. Chemical structure, porosity and thermostability of PUUMc which were obtained under various synthesis conditions were evaluated. Release rate of BaMa from PUUMc was slower and EI of BaMa was higher, when enzyme was encapsulated during PUUMc synthesis.

Phenolic compounds are the dominant pollutants in soils contaminated by the coking industry. Ring opening by the hydroxylase gene (bamA) is the key step in the benzoyl-CoA degradation pathway under anaerobic conditions, and a broad spectrum of microorganisms possesses this functional gene, including denitrifiers. The present study analyzed the community structure of denitrifying bacteria and the diversity of the bamA gene for mixed cultures enriched from soil collected at a coking industrial site and then grown under nitrate-reducing conditions on phenol or p-hydroxybenzoate (4HBA), a key intermediate product of anaerobic phenol degradation. Illumina sequencing of the 16S rRNA gene showed different bacterial compositions between the two cultures. The dominant phyla were Proteobacteria, Armatimonadetes, and Planctomycetes in the phenol culture and Proteobacteria and Bacteroidetes in the 4HBA culture. Phylogenetic analysis further demonstrated that bamA genes were associated with four clusters of bacteria, three of known bacteria and one of uncultured bacteria. The diversity of the bamA gene differed from that reported in anaerobic aromatic degradation cultures, suggesting that these enriched cultures may contain new strains unique to coking-contaminated soils. The present study further validates the potential application of this functional gene as a marker for anaerobic biodegradation processes in enrichment cultures from contaminated soil.

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

The aim of this study was to identify differentially expressed long non-coding RNAs (lncRNAs) molecules and predict their target genes related to muscle development and lipid metabolism in longissimus dorsi (LD) muscles of Bama Xiang pigs under constant heat stress. Ten male Bama Xiang pigs with an average initial body weight of 14 kg were randomly divided into control group (22°C) and heat stress (35 °C) group. The experiment lasted for 28 days. All the pigs were slaughtered at the end of the experiment, and LD muscles were collected for muscle quality analysis and transcriptome sequencing. Heat stress reduced meat quality of Bama Xiang pigs. lncRNAs in LD were identified systematically by deep RNA sequencing between the two groups. The results showed that 365 lncRNAs from the LD were identified, including 128 intergenic lncRNAs, 82 intronic lncRNAs, and 155 anti-sense lncRNAs. The differences lie in transcript of length, number of exons and wider size distribution, and expression level per KB fragment in three subtypes of lncRNAs. The three types of transposable elements coverage, including Line/L1, SINE/tRNA, and LTR/ERVL-MaLR, are the highest in mRNA and the three subtypes of lncRNAs in pigs. lncRNAs and mRNAs were different in comparison of features. The results predicted the target genes of the significant differentially expressed lncRNAs related to muscle development and lipid metabolism. This is the first study to expand the knowledge about muscle-related lncRNAs biology in Bama Xiang pigs under heat stress and will contribute to the development of alleviating the adverse effects of heat stress on pork quality targeting lncRNAs.

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.

Even though salbutamol (SAL) had remarkable effects on the enhancement of growth rate and carcass composition in different livestock species such as cattle, pigs, sheep and poultry, it was banned as a growth promoter because of its adverse effects on health. However, the specific mechanism by which salbutamol enhances growth efficiency remains unknown. In this study, Bama pigs were randomly allocated to receive salbutamol (5 mg/kg) for 30 or 60 days and were compared with untreated pigs. Pigs treated with salbutamol demonstrated enhanced growth rates and carcass composition; however, they showed deterioration in blood biochemical indices and organ development. We hypothesized that salbutamol exerts its effects by modulating the composition of the gut microbiota population. The faecal microbiome of pigs was characterized via pyrosequencing of the bacterial 16S rRNA gene. The gut microbiota population analysis showed that salbutamol caused shifts in the microbial composition of less abundant species. Redundancy analysis indicated an increase in abundance of the phylum Bacteroidetes, class Betaproteobacteria, family Christensenellaceae and genus Lactobacillus, and a decreased ratio of the phylum Firmicutes, class Clostridia and genera Ruminococcus, Blautia and Subdoligranulum. In conclusion, our study provided circumstantial evidence that the various effects of salbutamol are caused by gut microbiota modulation, and several potential candidates were identified for SAL detection via the gut microbiota. Our findings provided new insights into the roles of the gut microbiota during salbutamol treatment, and these findings will aid in the screening of alternative strategies for animal health improvement and production enhancement.

In Gram-negative bacteria, the biogenesis of β-barrel outer membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex. During the past decade, structural and functional studies have collectively contributed to advancing our understanding of the structure and function of the BAM complex; however, the exact mechanism that is involved remains elusive. In this Progress article, we discuss recent structural studies that have revealed that the accessory proteins may regulate essential unprecedented conformational changes in the core component BamA during function. We also detail the mechanistic insights that have been gained from structural data, mutagenesis studies and molecular dynamics simulations, and explore two emerging models for the BAM-mediated biogenesis of OMPs in bacteria.

Background Chinese Bama Yao Autonomous County is a well-known longevity region in the world. In the past 30 years, population and genome studies were undertaken to investigate the secret of longevity and showed that longevity is the result of a combination of multiple factors, such as genetic, environmental and other causes. In this study, characteristics of the blood plasma proteomic and autoantibody profiles of people from Bama longevity family were investigated. Methods Sixty-six plasma donors from Chinese Bama longevity area were recruited in this study. Thirty-three offsprings of longevous families were selected as case studies (Longevous group) and 33 ABO (blood type), age, and gender-matched subjects from non-longevous families were selected as controls (Normal group). Each group contains 3 biological replicates. Tandem mass tag-based proteomic technique was used to investigate the differentially expressed plasma proteins between the two groups. The auto-reactive IgG antibody profiles of the 3 pooled samples in each group were revealed by human proteome microarrays with 17,000 recombinant human proteins. Results Firstly, 525 plasma proteins were quantified and 12 proteins were discovered differentially expressed between the two groups. Secondly, more than 500 proteins were recognized by plasma antibodies, 14 proteins ware differentially reacted with the autoantibodies in the two groups. Bioinformatics analysis showed some of the differential proteins and targeted autoantigens were involved in cancer, cardiovascular disease and immunity. Conclusions Proteomic and autoantibody profiles varied between the offspring of longevous and normal families which are from the same area and shared the same environmental factors. The identified differences were reported to be involved in several physiological and pathological pathways. The identified proteins will contribute to a better understanding of the proteomic characteristics of people from Bama longevous area and a revelation of the molecular mechanisms of longevity. Electronic supplementary material The online version of this article (10.1186/s12014-019-9242-4) contains supplementary material, which is available to authorized users.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

β-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved β-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a β-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial β-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various β-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones.

Bama County (Guangxi, China) is famous for its longevous population. In this study, intestinal microflora of 17 healthy elderly subjects of different ages and from different regions (rural and urban) in Bama, were analyzed by denaturing gradient gel electrophoresis (DGGE). Significant effects of age and living region on the whole intestinal bacterial communities were observed by redundancy analysis (RDA). A total of 11 bacterial strains that were correlated with age and living region were identified using a t-value biplot combined with band sequencing. Four bacterial strains were correlated with both age and living region of the elderly in Bama. Two Bacteroides strains and one Ruminococcaceae strain were abundant in the rural, younger elderly; conversely, one Desulfovibrio strain was high in the urban, older elderly. Another Bacteroidetes strain was only correlated with the participant's age, and its abundance increased with the age of the elderly. The richness of one Clostridium sordellii strain, which was only correlated with the elderly living region, was high in the urban elderly. The study also found five other novel bacterial strains that were correlated with the age or living region of the elderly in Bama. These results expand our understanding of age- and region-effects on the intestinal microflora of the elderly and raise the possibility of developing probiotics originating from centenarians.

The phylogenetic relationship of five species of pigs including Guizhou miniature pig, Bama pig, Xisuangbanna inbred pig, Rongchang Pig and Landrance was studied by RAPD analysis. Thirty-nine single polymorphic primers were selected out of 190 primers. The amplified fragments of thirty-nine primers were analysed by the RAPDistance package version 1.04 and the phylogenetic tree was constructed using NJ method. The results indicated as the following: three strains of miniature pigs have close phylogenetic relationship and the phylogenetic relationship between Guizhou miniature pig and Bama miniature pig was much closer. Landrance and Rongchang pig have close phylogenetic relationship too, but their phylogenetic relationship is far from the three strains of miniature pigs.

Intestinal microflora analysis was performed on 52 healthy elderly subjects of different ages and in different regions in Bama County, Guangxi, China. The participants were assigned to three groups depending on their age and location: longevous (group M; mean age = 98 years; n = 21); rural younger elderly (group S; mean age = 70 years; n = 18); and urban elderly (group C; mean age = 82 years; n = 13). Ten groups of bacteria were quantified using real-time PCR. Age-related differences were observed in the number of Clostridium coccoides-Eubacterium rectale--there were more in longevous participants. Region affected the numbers of Bacteroides--Prevotella and Clostridium perfringens subgroup, and longevous participants had significantly more of the two bacterial groups than urban elderly participants. Region-related effects were also observed for the relative abundance of E. coli, and rural elderly participants had a lower proportion. Both age and regional effects were observed in the amount of total bacteria, and longevous participants had higher numbers than urban elderly participants. A significantly higher proportion of lactobacilli was observed in rural younger elderly participants than urban elderly participants, but independent age or regional effects did not contribute to this difference. This study suggests that age and region can affect the intestinal microflora of elderly people.

Four genes (bamA1, bamA2, bamA3 and bamA4) encoding homologues of the gamma-butyrolactone autoregulator receptor of Streptomyces were found and cloned from Amycolatopsis mediterranei, a typical non-Streptomyces actinomycetes and a producer of rifamycin, one of the major anti-tuberculosis drugs in clinical treatment. Transcriptional analysis demonstrated that bamA1 and bamA2 are transcribed in a growth-dependent manner, while bamA3 and bamA4 are constitutively transcribed during growth. Binding assays using (3)H-labeled autoregulator analogues as ligands confirmed that all of the recombinant BamA proteins expressed in Escherichia coli have clear binding activity toward several types of Streptomyces autoregulators. The ligand specificity of the recombinant BamA1 protein was identical to that of the crude cell-free lysates of A. mediterranei reported in our previous work. These results suggest that A. mediterranei, which is phylogenetically situated in a distal clade from the genus Streptomyces as non-Streptomyces actinomycetes, has an autoregulator-mediated signaling system.

Background Like all diderm bacteria studied to date, Borrelia burgdorferi possesses a β-barrel assembly machine (BAM) complex. The bacterial BAM complexes characterized thus far consist of an essential integral outer membrane protein designated BamA and one or more accessory proteins. The accessory proteins are typically lipid-modified proteins anchored to the inner leaflet of the outer membrane through their lipid moieties. We previously identified and characterized the B. burgdorferi BamA protein in detail and more recently identified two lipoproteins encoded by open reading frames bb0324 and bb0028 that associate with the borrelial BamA protein. The role(s) of the BAM accessory lipoproteins in B. burgdorferi is currently unknown. Results Structural modeling of B. burgdorferi BB0028 revealed a distinct β-propeller fold similar to the known structure for the E. coli BAM accessory lipoprotein BamB. Additionally, the structural model for BB0324 was highly similar to the known structure of BamD, which is consistent with the prior finding that BB0324 contains tetratricopeptide repeat regions similar to other BamD orthologs. Consistent with BB0028 and BB0324 being BAM accessory lipoproteins, mutants lacking expression of each protein were found to exhibit altered membrane permeability and enhanced sensitivity to various antimicrobials. Additionally, BB0028 mutants also exhibited significantly impaired in vitro growth. Finally, immunoprecipitation experiments revealed that BB0028 and BB0324 each interact specifically and independently with BamA to form the BAM complex in B. burgdorferi. Conclusions Combined structural studies, functional assays, and co-immunoprecipitation experiments confirmed that BB0028 and BB0324 are the respective BamB and BamD orthologs in B. burgdorferi, and are important in membrane integrity and/or outer membrane protein localization. The borrelial BamB and BamD proteins both interact specifically and independently with BamA to form a tripartite BAM complex in B. burgdorferi. A working model has been developed to further analyze outer membrane biogenesis and outer membrane protein transport in this pathogenic spirochete.

Summary β-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features speak to the mechanism by which BamA catalyzes β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, evocatively suggesting a route from the interior cavity in BamA into the outer membrane.

TamA is an Escherichia coli Omp85 protein involved in autotransporter biogenesis. It comprises a 16-stranded transmembrane β-barrel and three POTRA domains. The 2.3-Å crystal structure reveals that the TamA barrel is closed at the extracellular face by a conserved lid loop. The C-terminal β-strand of the barrel forms an unusual inward kink, which weakens the lateral barrel wall and creates a gate for substrate access to the lipid bilayer.