Autodisplay of streptavidin

Streptavidin was expressed on the outer membrane of E. coli as a recombinant fusion protein with an autotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technology. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single E. coli cell was evaluated with densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment with fluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activity of the E. coli with autodisplayed streptavidin was compared with the activity of streptavidin immobilized on magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and layered on a 96-well microplate for an immunoassay.     

Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains

“Autodisplay technology” is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.     

A magnetite suspension-based washing method for immunoassays using Escherichia coli cells with autodisplayed Z-domains

Escherichia coli cells with autodisplayed Z-domains have been used for immunoassays of specific target analytes. In this study, a magnetite suspension was used for the washing step in immunoassays of E. coli cells with autodisplayed Z-domains. This approach enhanced the washing conditions for these immunoassays by determining (1) the optimal concentration of the magnetite suspension, (2) the capacity of the magnetite suspension-based washing method to recover E. coli cells, and (3) the level at which the activity of autodisplayed Z-domains is maintained. In immunoassays of C-reactive protein (CRP), the immunoassay incorporating the magnetite suspension-based washing method showed a sensitivity and limit of detection considerably higher than those of the conventional centrifugation-based washing method. The results indicated that immunoassays incorporating the magnetite suspension-based washing method are effective for medical diagnoses based on CRP assay.     

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.     

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation.Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.     

The C-terminal amphipathic α-helix of Pseudomonas aeruginosa PelC outer membrane protein is required for its function

Pseudomonas aeruginosa is an opportunistic pathogen, which causes numerous infections and can adopt a versatile lifestyle. During chronic infection, P. aeruginosa becomes established as a bacterial community known as a biofilm. Biofilm formation results from the production of a matrix mainly comprised of exopolysaccharides. P. aeruginosa possesses several gene clusters which contribute to the formation of the matrix, including the pel genes. Among the pel genes, pelC encodes an outer membrane protein, which may serve as a transporter of polysaccharide to the bacterial cell surface. Whereas outer membrane proteins usually display an amphipathic β-barrel fold, we show that PelC requires a C-terminal amphipathic α-helix for outer membrane insertion and function. Such a structural feature has only previously been reported for the Wza outer membrane protein of Escherichia coli, and our data suggest that this characteristic may be found in a large family of proteins, particularly outer membrane proteins specialized in polysaccharide transport.     

Crystal Structures of CusC Review Conformational Changes Accompanying Folding and Transmembrane Channel Formation

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance–nodulation–cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel.     

The Regulated Outer Membrane Protein Omp21 from Comamonas acidovorans Is Identified as a Member of a New Family of Eight-Stranded β-Sheet Proteins by Its Sequence and Properties

Omp21, a minor outer membrane protein of the soil bacterium Comamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of β structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic β strands, which includes virulence proteins, such as the neisserial opacity proteins, Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF from Pseudomonas aeruginosa.     

FORMATION AND STRUCTURE OF THE SPORE OF BACILLUS COAGULANS

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.     

Fine Structure of Thermus aquaticus, an Extreme Thermophile

Electron microscopic studies using thin sections revealed that Thermus aquaticus has a structure similar to that of most other gram-negative bacteria. The cell envelope is tripartite: plasma membrane, thin middle layer, and a thicker and irregular outer layer. The outer layer appears to be joined to the plasma membrane by a series of connections and, when seen in tangential section, the outer layer appears as a series of parallel bands. The cell division mechanism resembles that of typical gram-negative bacteria. Large spherical bodies designated “rotund bodies” are formed as a result of the association of a number of separate cells. In this association the outer envelope layers of the cells fuse and pull away from the middle layer. The rotund body thus appears as a series of rods, usually lying in parallel around the periphery of the sphere, completely connected by means of the fused outer layer.     

Description of Complex Forms of a Porin in Bacteroides fragilis and Possible Implication of this Protein in Antibiotic Resistance

Periodic surveys of antibiotic susceptibility patterns among anaerobes have emphasized that new mechanisms of resistance have emerged, especially in the Bacteroides fragilis group. Resistance to the combination of amoxicillin and clavulanic acid among some imipenem-susceptible Bacteroides fragilis strains has been associated with modifications in outer membrane protein electrophoretic patterns with the loss of some porin-like proteins. Porins are outer membrane proteins that play a major part in membrane permeability; if they are under-expressed, they can be responsible for antibiotic resistance. In a previous work, we isolated one outer membrane protein of 45 kDa from Bacteroides fragilis and showed its porin activity. In the present study, we aim to isolate the different complex forms of this protein and to underline their possible role in antibiotic resistance. We therefore compared the electrophoretic patterns of the outer membrane proteins of several strains of Bacteroides fragilis. Although these patterns are similar to each other, some proteins, especially those of high molecular weight, are less visible in the samples heated before electrophoresis. We targeted these high molecular weight proteins (which appeared sensitive to heat) and isolated them by electro-elution. We thus identified two high molecular weight proteins (210 and 130/135 kDa) which seemed to be components of a complex including the 45 kDa outer membrane protein formerly identified by us as a porin protein. Their porin activities were tested by the swelling assay of proteoliposomes which showed that the 210 kDa protein behaved like the 45 kDa protein whereas the 130/135 kDa protein had less porin activity. Furthermore, swelling assays with antibiotic solutions made it possible to compute the role of this protein complex in antibiotic resistance.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Translocation and assembly of outer membrance proteins of Escherichia coli. Selective accumulation of precursors and novel assembly intermediates caused by phenethyl alcohol.

Selective and step-wise inhibition of bioysnthesis and assembly of three major outer membrane proteins of Escherichia coli (matrix protein, tolG protein (DiRienzo et al., 1978), and lipoprotein) was achieved in the presence of phenethyl alcohol. At a lower concentration (0·3% or higher) PEA4 specifically inhibited the processing and assembly of matrix protein, resulting in the accumulation of promatrix protein. The promatrix protein thus synthesized in the presence of PEA was chased into matrix protein and properly assembled into the outer membrane upon the removal of PEA, demonstrating a direct precursor-product relationship between the two proteins. Promatrix protein was sensitive to trypsin and was also solubilized from the membrane fraction by sodium sarcosinate. However, promatrix protein was also found to be loosely associated with the outer membrane fraction. These data indicate that promatrix protein was translocated across the cytoplasmic membrane and localized external to the cytoplasmic membrane, although it was not yet properly inserted into the outer membrane structure.The inhibition of processing of protolG (DiRienzo et al., 1978) protein was observed at higher levels of PEA (0·4% or higher). However, at all concentrations of PEA tested, the accumulation of prolipoprotein was not detected. On the other hand, when PEA was added at concentrations lower than the above critical concentrations for each protein, the precursor was properly processed but the processed proteins (tolG protein, and lipoprotein) were accumulated in the periplasmic space, since they were released by osmotic shock. tolG protein of the soluble cell fraction was chased into the outer membrane after removal of PEA and regrowth of the cells in culture. The processed lipoprotein of the soluble fraction was trypsin-sensitive in contrast to mature lipoprotein. These results indicate that the precursor protein with the peptide extension is transformed into a new assembly intermediate after the extended peptide is cleaved off. This intermediate may be released into the periplasmic space in the presence of PEA before it can be assembled into the outer membrane. These data indicate that the peptide extension is not essential for the insertion of the outer membrane protein into the outer membrane.When PEA (0·3%) was added to a growing culture, the production of not only matrix protein but also promatrix protein was completely inhibited. However, synthesis of promatrix protein was restored when rifampicin was added before the PEA treatment. These results are discussed in terms of control of gene expression for matrix protein. PEA was found to increase the membrane fluidity.     

Integration and Oligomerization of Bax Protein in Lipid Bilayers Characterized by Single Molecule Fluorescence Study

Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-x_L. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-x_L can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis.     

Collagen-Like Proteins (ClpA,ClpB, ClpC,and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42

The genes of collagen-like proteins (CLPs) have been identified in a broad range of bacteria, including some human pathogens. They are important for biofilm formation and bacterial adhesion to host cells in some human pathogenic bacteria, including several Bacillus spp. strains. Interestingly, some bacterial CLP-encoding genes (clps) have also been found in non-human pathogenic strains such as B. cereus and B. amyloliquefaciens, which are types of plant-growth promoting rhizobacteria (PGPR). In this study, we investigated a putative cluster of clps in B. amyloliquefaciens strain FZB42 and a collagen-related structural motif containing glycine-X-threonine repeats was found in the genes RBAM_007740, RBAM_007750, RBAM_007760, and RBAM_007770. Interestingly, biofilm formation was disrupted when these genes were inactivated separately. Scanning electron microscopy and hydrophobicity value detection were used to assess the bacterial cell shape morphology and cell surface architecture of clps mutant cells. The results showed that the CLPs appeared to have roles in bacterial autoaggregation, as well as adherence to the surface of abiotic materials and the roots of Arabidopsis thaliana. Thus, we suggest that the CLPs located in the outer layer of the bacterial cell (including the cell wall, outer membrane, flagella, or other associated structures) play important roles in biofilm formation and bacteria-plant interactions. This is the first study to analyze the function of a collagen-like motif-containing protein in a PGPR bacterium. Knocking out each clp gene produced distinctive morphological phenotypes, which demonstrated that each product may play specific roles in biofilm formation. Our in silico analysis suggested that these four tandemly ranked genes might not belong to an operon, but further studies are required at the molecular level to test this hypothesis. These results provide insights into the functions of clps during interactions between bacteria and plants.     

The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.     

Microbial population dynamics and proteomics in membrane bioreactors with enzymatic quorum quenching

Quorum sensing gives rise to biofilm formation on the membrane surface, which in turn causes a loss of water permeability in membrane bioreactors (MBRs) for wastewater treatment. Enzymatic quorum quenching was reported to successfully inhibit the formation of biofilm in MBRs through the decomposition of signal molecules, N-acyl homoserine lactones (AHLs). The aim of this study was to elucidate the mechanisms of quorum quenching in more detail in terms of microbial population dynamics and proteomics. Microbial communities in MBRs with and without a quorum quenching enzyme (acylase) were analyzed using pyrosequencing and compared with each other. In the quorum quenching MBR, the rate of transmembrane pressure (TMP) rise-up was delayed substantially, and the proportion of quorum sensing bacteria with AHL-like autoinducers (such as Enterobacter, Pseudomonas, and Acinetobacter) also decreased in the entire microbial community of mature biofilm in comparison to that in the control MBR. These factors were attributed to the lower production of extracellular polymeric substances (EPS), which are known to play a key role in the formation of biofilm. Proteomic analysis using the Enterobacter cancerogenus strain ATCC 35316 demonstrates the possible depression of protein expression related to microbial attachments to solid surfaces (outer membrane protein, flagellin) and the agglomeration of microorganisms (ATP synthase beta subunit) with the enzymatic quorum quenching. It has been argued that changes in the microbial population, EPS and proteins via enzymatic quorum quenching could inhibit the formation of biofilm, resulting in less biofouling in the quorum quenching MBR.     

Synthesis and turnover of the regularly arranged surface protein of Acinetobacter sp. relative to the other components of the cell envelope.

The formation of the components of the cell envelope of Acinetobacter sp. 199A was investigated by measuring the incorporation of [3H]leucine into protein, [14C]galactose into lipopolysaccharide, 32P into phospholipid, and [3H]diaminopimelic acid into peptidoglycan. Whereas the lipopolysaccharide and intrinsic protein of the outer membrane were stable, some of the regularly arranged surface protein, the alpha-protein, was lost into the growth medium. Only newly synthesized alpha-protein was lost. The peptidoglycan of the murein layer was also labile. Selective inhibition of the formation of individual components of the cell envelope with penicillin, chloramphenicol, and bacitracin showed that incorporation of protein into the outer membrane required the simultaneous formation of complete lipopolysaccharide. The converse was not true: protein synthesis was not required for lipopolysaccharide incorporation. Formation of the outer membrane and the murein layer proceeded independently.