Role of a two-component ResD-ResE system in regulating the expression of guanyl-specific ribonuclease genes in <Emphasis Type="Italic">Bacilli</Emphasis>

The role of the two-component ResD-ResE signal transduction system in regulating the expression of guanyl-specific ribonuclease genes in bacilli has been studied. Proteins with homologies to the ResD and ResE regulatory proteins of Bacillus subtilis have been found in all sequenced genomes of Bacillus. It has been shown using the B. subtilis strains defective in genes of these proteins that the ResD-ResE signal transduction system positively regulates the expression of ribonuclease genes of B. intermedius, B. pumilus, and B. thuringiensis in cells of B. subtilis. The data obtained in this work speak for the fact that regulatory system similar to the two-component ResD-ResE signal transduction system of B. subtilis also functions in other representatives of the Bacillus genus.     

Chaperone-protease systems in regulation and protein quality control in Bacillus subtilis

The Gram-positive model organism Bacillus subtilis is extremely well adapted to changing environmental conditions. The chaperone-protease ClpCP and other AAA+ proteases constitute an important component of the B. subtilis protein quality control system that is essential for survival during stress. In this review, we discuss recent discoveries concerning the molecular mechanism, regulation and localization of proteases and chaperones in B. subtilis.     

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480 mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (P_fen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the P_pps promoter from B. subtilis BBG111 was replaced by promoter P_fen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with P_fen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.     

The “Protein Corona” of Silver-Sulfide Nanoparticles Obtained Using Gram-Negative and -Positive Bacteria

A comparative analysis of the amount and composition of the proteins adsorbed onto the surface of silver-sulfide (NpAg₂S) nanoparticles obtained by biosynthesis using bacteria was carried out for the first time. These were the gram-negative bacteria Shewanella oneidensis MR-1 and Escherichia coli K12, as well as the gram-positive bacterium Bacillus subtilis 168. The biosynthesis of NpAg₂S was carried out in nutrient broth with 1 mM of AgNO₃ and Na₂S₂O₃ · 5H₂O salts in the presence of bacterial cells under aerobic conditions. Analysis of NpAg₂S by transmission electron microscopy showed that the particles were spherical and had an average diameter of 8 ± 2 nm for S. oneidensis MR-1 and E. coli K12 and 10 ± 3 nm for B. subtilis 168. It was found that the highest amount of protein was sorbed on NpAg₂S, when the strain B. subtilis 168 was used, and the smallest amount was sorbed by E. coli. The main proteins adsorbed on NpAg₂S were determined by the MALDI TOF/TOF method, and the heterogeneity of the protein coating was revealed. The lowest heterogeneity of proteins on the surface of nanoparticles is observed in the case of B. subtilis (only one protein, flagellin, predominates); the highest heterogeneity of proteins was found on nanoparticles obtained using S. oneidensis MR-1. All proteins covering the surface of NpAg₂S were shown to be outer-membrane or cytoplasmic-membrane proteins of the studied bacteria. The composition of the protein coating of nanoparticles is individual and constant for each bacterial strain used. The values of the ζ-potential and the effective diameter of nanoparticles were shown to differ depending on the “protein corona” of the strain that was used to obtain NpAg₂S. The characteristic of the “protein corona” of biologically obtained nanoparticles is an important and necessary condition for their practical application.     

Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis

Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa_247-370 polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa_247-370 on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa_247-370-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.     

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.     

Heat-killed Bacillus subtilis inhibits T-cell proliferative response to mitogens and recall antigens

Heat-killed vegetative forms of Bacillus subtilis were found to impair considerably the capacity of human T-lymphocytes to secrete interleukin-2 (IL-2) and to proliferate (in terms of [³H]thymidine incorporation) after phytohaemagglutinin (PHA) stimulation. B. subtilis was also found to interfere with T-cell proliferation induced by concanavalin A (Con A) and the recall antigen tetanus toxoid (TT). The suppressive activity was dependent on bacterial concentration, and was not ascribed to mitogen, medium-nutrient absorption or cell killing. Moreover, B. subtilis did not interfere with mitogen-induced IL-2 receptor expression on the T-cell surface. On the other hand, B. subtilis did not interfere with T-cell proliferation induced by phorbol myristate acetate (PMA) and ionomycin stimulation. All data obtained suggest the binding of B. subtilis subcomponents to — or very close to — the T-cell receptor (TCR). Identification and purification of the basic structure(s) or component(s) of B. subtilis with TCR antagonist activity in vitro will help to exploit different aspects of T-cell activity and development, and possibly, will provide a means of specific control or modification of the immune response.     

Epigenetic regulation of olfactory receptor gene expression by the Myb–MuvB/dREAM complex

In both mammals and insects, an olfactory neuron will usually select a single olfactory receptor and repress remaining members of large receptor families. Here we show that a conserved multiprotein complex, Myb–MuvB (MMB)/dREAM, plays an important role in mediating neuron-specific expression of the carbon dioxide (CO₂) receptor genes (Gr63a/Gr21a) in Drosophila. Activity of Myb in the complex is required for expression of Gr63a/Gr21a and acts in opposition to the histone methyltransferase Su(var)3-9. Consistent with this, we observed repressive dimethylated H3K9 modifications at the receptor gene loci, suggesting a mechanism for silencing receptor gene expression. Conversely, other complex members, Mip120 (Myb-interacting protein 120) and E2F2, are required for repression of Gr63a in inappropriate neurons. Misexpression in mutants is accompanied by an increase in the H3K4me3 mark of active chromatin at the receptor gene locus. Nuclei of CO₂ receptor-expressing neurons contain reduced levels of the repressive subunit Mip120 compared with surrounding neurons and increased levels of Myb, suggesting that activity of the complex can be regulated in a cell-specific manner. Our evidence suggests a model in which olfactory receptors are regulated epigenetically and the MMB/dREAM complex plays a critical role in specifying, maintaining, and modulating the receptor-to-neuron map.     

Bacillus subtilis isolated from the human gastrointestinal tract

As part of an ongoing study to determine the true habitat of Bacillus species, we report here the isolation and characterisation of Bacillus subtilis from the human gastrointestinal tract (GIT). Strains were obtained from ileum biopsies as well as from faecal samples and their biotypes defined. 16S rRNA analysis revealed that most isolates of B. subtilis were highly conserved, in contrast to RAPD-PCR fingerprinting that showed greater diversity with 23 distinct RAPD types. The majority of B. subtilis strains examined possessed features that could be advantageous to survival within the GIT. This included the ability to form biofilms, to sporulate anaerobically and secretion of antimicrobials. At least one isolate was shown to form spores that carried an exosporium, a loosely attached outer layer to the mature endospore, this being the first report of B. subtilis spores carrying an exosporium. This study reinforces a growing view that B. subtilis and probably other species have adapted to life within the GIT and should be considered gut commensals rather than solely soil microorganisms.     

The Brucella suis type IV secretion system assembles in the cell envelope of the heterologous host Agrobacterium tumefaciens and increases IncQ plasmid pLS1 recipient competence

Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α₂-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.     

Heterologous production of the P1 porin of Neisseria meningitidis in Bacillus subtilis: the effect of an N-terminal extension on the presentation of native-like epitopes

The major outer membrane protein P1 (class 1) ofNeisseria meningitidishas been produced as inclusion bodies inBacillus subtiliswith the aim to develop a vaccine based on it. The protein produced in high yield inB. subtiliscontained an N-terminal extension of 11 amino acid residues which was found to be necessary for expression in the production system. In the present study we asked whether or not the removal of this extension would effect the conformation of this protein in liposomes as judged by its immunogenic properties. A methionine was engineered in front of the mature P1 protein to provide a chemical cleavage site for CNBr to remove the extension. The CNBr-cleaved protein, complexed with phospholipids, elicited high titers of antibodies binding to the meningococcal cells similarly to the noncleaved protein. This suggests that the BacP1 protein can serve as an effective vaccine component irrespective of the presence, or absence, of this N-terminal extension.     

Protein A as a molecular probe for the detection of antigen induced conformational change in Fc region of rabbit antibody

Rabbit IgG antibody which reacts with protein A of Staphylococcus aureus (SpA) and forms a soluble complex with molar composition (IgG₂-SpA₁)₂ is not able to further bind SpA or to attach to SpA-Sepharose 4B thus proving that the unengaged SpA reactive sites of IgG are shielded by the already bound SpA.On the contrary the (IgG₂-SpA₁)₂ complex was able to bind a small fragment of SpA (fSpA) clearly showing that SpA-reactive sites are present in the rabbit IgG molecules of the complex but that they are not available for the intact SpA molecule.Immune complexes contaning (IgGaFER₂-SpA₁)₂ and ferritin attach to an SpA-Sepharose 4B column showing that SpA binding sites exist on the IgG molecules and became exposed after the conformational change of the Fcγ region induced by the antigen. Therefore SpA can be used for the direct detection of conformational changes induced by antigen in the Fcγ region of rabbit antibody.     

Detection of Bacillus anthracis spore germination in vivo by bioluminescence imaging

We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.     

SwrA as global modulator of the two-component system DegSU in Bacillus subtilis

The two-component system DegSU of Bacillus subtilis controls more than one hundred genes involved in several different cellular behaviours. Over the last four decades, the degU32^Hy allele, supposedly encoding a constitutively active mutant of the response regulator DegU, was exploited to define the impact of this system on cell physiology. Those studies concluded that phosphorylated DegU (DegU∼P) induced degradative enzyme expression while repressing flagellar motility and competence.Recent experiments, however, demonstrated that flagella expression is enhanced by DegU∼P if SwrA, a protein only encoded by wild strains, is present. Yet, to promote motility, SwrA must interact with DegU∼P produced by a wild-type degU allele, as it cannot correctly cooperate with the mutant DegU32^Hy protein.In this work, the impact of DegSU was reanalysed in the presence or absence of SwrA employing a DegS kinase mutant, degS200^Hy, to force the activation of the TCS. Our results demonstrate that the role of SwrA in B. subtilis physiology is wider than expected and affects several other DegSU targets. SwrA reduces subtilisin, cellulases and xylanases production while, besides motility, it also positively modulates competence for DNA uptake, remarkably relieving the inhibition caused by DegU∼P alone and restoring transformability in degS200^Hy strains.     

Wingless signaling regulates winner/loser status in Minute cell competition

Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild‐type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, we established two assay systems for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3‐RNAi‐expressing cells) system in which RpS3‐RNAi‐expressing wing pouch cells are eliminated from wild‐type wing disc and (ii) the SURE (supercompetition of RpS3‐expressing clones in RpS3/+ tissue) system in which RpS3‐over‐expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over‐expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down‐regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, we found that down‐regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. Our observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition.     

Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae

Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3). Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O₁ ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F’ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.     

Hypoxic response elements and Tet-On advanced double-controlled systems regulate hVEGF165 and angiopoietin-1 gene expression in vitro

Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic re-sponse elements (HRE) and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF165 and angiopoietin-1 (Ang-1) genes in rat cardiomyocytes exposed to hypoxia and pharma-cologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE- hVEGF165. Our results indicated that the viral titer was 1×1012 vg /mL and the viral purity exceeded 98%. hVEGF165 expression was induced by hypoxia, but not by normoxia (P < 0.001). Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction (P < 0.001). Immunofluorescence staining showed that positively stained hVEGF165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.