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1.
A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.  相似文献   

2.
Summary Overproduction of extracellular endoglucanase was attempted by modifying promoter region of an endoglucanase gene cloned from Bacillus subtilis BSE616 and expressing in B. subtilis DB104. A strong promoter was cloned from B. subtilis 168 chromosomal DNA and fused to the endoglucanase gene after removing its native promoter. An effective Shine-Dalgarno sequence was inserted between the promoter and the endoglucanase structural gene. The modified gene was expressed well in B. subtilis and produced 265 units of endoglucanase per mg protein that is 60 % of total protein which was secreted into culture medium.  相似文献   

3.
FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.  相似文献   

4.
5.
YlyA是枯草杆菌一种功能未知蛋白.本研究旨在建立可溶性YlyA的诱导表达体系和纯化方法,为其功能研究奠定基础.PCR扩增ylyA序列,将其克隆到pETMCSⅢ中构建表达载体pNG252,用IPTG诱导6×His-YlyA融合蛋白在大肠杆菌BL21(DE3)中表达,对表达产物进行分析,最后对可溶性YlyA重组蛋白进行Ni2+-WTA亲和层析加以纯化.结果表明pNG252中ylyA的插入方向正确,序列无突变;用0.5 mmol/L IPTG,37℃诱导3 h时,YlyA虽高效表达,但为包涵体形式;调整诱导条件至0.05 mmoL/L IPTG,25℃,5 h时,高效表达的YlyA部分转为可溶性蛋白.纯化后的YlyA浓度达204.2119 μmoL/L;调整洗涤液中的咪唑浓度至15 mmol/L,pH至9,可使纯化蛋白的产量大幅提高.本文成功构建了YlyA高效可诱导表达载体,建立了可溶性蛋白的诱导表达条件,确立了Ni2+-NTA亲和层析纯化方法,所得的6×His-YlyA融合蛋白,可用于YlyA晶体结构和与功能分析.  相似文献   

6.
The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.  相似文献   

7.
Bacillus subtilis XF-1 has been used as a biocontrol agent of clubroot disease of crucifers infected by Plasmodiophora brassicae, an obligate pathogen. In order to maximize the growth inhibition of the pathogen, random mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine was applied to strain XF-1. The efficacy of 226 selected mutants was assessed against the growth of an indicator fungal pathogen: Fusarium solani using agar plate assay and the disruptive effects on the resting spores of P. brassicae. Four mutants exhibited inhibition activity significantly higher than the wild type. The cell extracts of these mutants and the XF-1 were subjected to matrix-assisted laser desorption ionization-time of flight mass spectra analysis, and three families of cyclic lipopeptides (CLPs) fengycin, surfactin and iturin were identified from the parental strain and the screened mutants. However, the relative contents and compound diversity changed after mutagenesis, and there was slight variation in the surfactin and fengycin. Notably, only 5 iturin components were discovered from the wild strain XF-1, but 13 were obtained from the mutant strains, and the relative CLPs contents of all mutant strains increased substantially. The results suggested that CLPs might be one of main biocontrol mechanisms of the clubroot disease by XF-1. The 4 mutants are far more effective than the parental strain, and they would be promising biocontrol candidates not only against P. brassicae but probably other plant diseases caused by fungi.  相似文献   

8.
A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.  相似文献   

9.
Bacillus subtilis strains isolated from cowdung (CD) had several beneficial attributes, which included biocontrol, plant growth promotion, sulphur (S) oxidation, phosphorus (P) solubilization and production of industrially important enzymes (amylase and cellulase). The B. subtilis strains from CD inhibited the in vitro growth of fungi, Fusarium oxysporum (25-34%) and Botryodiplodia theobromae (100%), isolated from the postharvest rots of yam (Dioscorea rotundata) tubers. Other than biocontrol, B. subtilis strains were able to promote root elongation in seedlings of Cicer arietinum up to 70-74% as compared to untreated seeds (control). B. subtilis strains had also the ability to oxidize elemental S to sulphate (2-15microgml(-1)) and showed distinct P-solubilization activity in vitro. In addition, the cultures showed cellulase activity in carboxy methyl cellulose medium (1.5-1.8mg of reducing sugar24h(-1)ml(-1)) and amylase activity in vitro.  相似文献   

10.
Muskmelon (Cucumis melo L.) wilt caused by Fusarium oxysporum f. sp. melonis leads to severe economic losses. A bio-organic fertilizer (BIO) fortified with an antagonistic strain of Bacillus subtilis Y-IVI was used to control this disease. Pot experiments were carried out to investigate the efficacy and to elucidate biocontrol mechanisms for the disease. BIO significantly reduced the disease incidence. Population of F. oxysporum in plant shoots of the BIO treatment were about 1000-fold lower than the control. Population of Y-IVI remained high in muskmelon rhizosphere of the BIO treatment during the experiment. Concentration of antifungal lipopeptides, iturin A, in the BIO treatment was significantly higher than other treatments. Ten days after transplantation, the salicylic acid content in BIO-treated plant leaves was significantly higher than control. In conclusion, BIO effectively controlled muskmelon wilt, possibly because the antagonistic microbes effectively colonize the plant rhizosphere and shoots to preclude pathogen invasion. Furthermore, Y-IVI produces antifungal lipopeptides in the rhizosphere.  相似文献   

11.
12.
Bacillus thuringiensis (Bt) is a microbial pesticide widely used to control crop pests. Its strains have good biocontrol activity against crop insect pest, but lack some desirable characteristics that are found in Bacillus subtilis. An attempt has been made to combine those desirable characteristics; we used a highly effective biocontrol strain of B. thuringiensis in protoplast fusions with a strain of B. subtilis. The fusants were identified through cell culture and stained with crystal violet. The Bt and B. subtilis protoplasts were induced to fuse by PEG 6000. The fusants were produced almost 95% mortality in first instar larvae of Spodoptera litura. The lethal doses (The LC50 and LC90) for mortality of S. litura values were significantly in lower level in the fusant-treated larvae, when compared with Bt and B. subtilis individual treatment. The consumption and digestion of S. litura significantly decreased after treatment with fusant. Also the approximate digestibility of S. litura increased significantly.  相似文献   

13.
High-level production (880 mg liter−1) and isolation of the anteiso-C17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.The soil bacterium Bacillus subtilis ATCC 6633 synthesizes the lipopeptides mycosubtilin and surfactin via a so-called nonribosomal peptide synthetase. Mycosubtilin belongs to the iturin family and is composed of seven α-amino acids linked to a unique C16 or C17 β-amino fatty acid with a linear or branched (iso or anteiso) acyl chain (15). This amphiphilic structure confers interesting biological properties on this secondary metabolite, particularly antifungal activity, which increases with the number of carbon atoms of the acyl chain (10). However, studies and applications of mycosubtilin are compromised by limited production by the native producer, cosynthesis of surfactin, and the existence of different mycosubtilin homologues and isoforms. In this work, utilization of specific precursors together with appropriate culture conditions for a genetically engineered strain led to the synthesis of a large amount of the most biologically active mycosubtilin homologue. Structural characterizations by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy demonstrated that this homologue corresponds to the anteiso-C17 isoform. Its antifungal activity against pathogenic Candida spp. was examined by MIC determination, fluorescence spectroscopy, and leakage experiments.Overproduction of mycosubtilin in B. subtilis ATCC 6633 was obtained by replacement of the native promoter of the myc operon, which encodes mycosubtilin synthetase, by the strong and tightly regulated xylA promoter from the Bacillus megaterium xylose isomerase (16). To this end, a repressor-promoter xylR-pxylA fragment was PCR amplified from pAXO1 (9) with Pfu DNA polymerase (Promega) and primers R100 and R101 before being cloned at the HincII site of pBG103 to yield pBG113 (Tables (Tables11 and and2).2). Then, a spectinomycin resistance cassette was rescued from pRFB122 by PstI/EagI digestion and inserted into pBG113 at the corresponding site to yield pBG113s (Table (Table1).1). This construct was used to transform B. subtilis ATCC 6633 as previously described (8), and transformants were selected on Luria-Bertani plates supplemented with spectinomycin (100 μg/ml). Correct integration in the resulting RFB107 strain was verified by analytical PCR using primers R102 and R103 (Table (Table2).2). In a second step, the srf operon, encoding surfactin synthetase, was disrupted in RFB107 to render the strain unable to synthesize surfactin. The knockout was targeted downstream of the comS regulator involved in competence mechanisms, which lies nested and out of frame within the srf operon (6, 7). The disruption cassette was obtained by a ligation-mediated PCR method (12) as follows. Fragments of srfAB and srfAD open reading frames were PCR amplified (primer pairs R106 and R107 and R108 and R109, respectively) from B. subtilis ATCC 6633 genomic DNA (obtained with the Wizard genomic DNA purification kit; Promega) while the erythromycin resistance cassette (ERY) of pDML1567 was amplified with primers R110 and R111. PCR fragments (200 ng) were digested with SfiI, purified with the GFX purification kit (GE Healthcare), and ligated with T4 DNA ligase (Promega) before being used as a template for PCR amplification using primers R106 and R109. The resulting srfAB-ERY-srfAD cassette was then used to transform B. subtilis RFB107, and transformants were selected on Luria-Bertani erythromycin (1 μg/ml) plates. Integration by a double-crossing event in the resulting strain RFB112 was verified by analytical PCR using primers R104 and R105 (Table (Table1)1) and by liquid chromatography-electrospray ionization-mass spectrometry analysis of the purified culture supernatant as described elsewhere (14).

TABLE 1.

Plasmids and Bacillus strains used in this study
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TABLE 2.

Synthetic primers used for PCR amplification
NameSequencea (5′-3′)Restriction site
R100GGGAGCTCGGATCCCATTTCCC
R101CGATATCTCTGCAGTCGCGATG
R102CCACTCCTTTGTTTATCCACCGAAC
R103GACGTTCAAATAAGTGTGATTGGCC
R104ACGGAGGGAGACGATTTGCA
R105ACATTCGGTGAATAAGGAAGCA
R106GTGAAAATCCGAGGCTACCGC
R107GGGGGCCCCAGCGGCCATATAAGCCGCCAGCTGGCGSfiI
R108CAGGGCCCAGTGGGCCGCAGGGCGAAACGCTAGATAGGSfiI
R109GCTGTCACAAACGGAAGAAGTC
R110CATGGCCCACTGGGCCCTGCTTCCTAATGCAGGAGTCGCSfiI
R111CCCCGGCCGCTGGGGCCCCCGCGATCGCCTATTTGGCSfiI
Open in a separate windowaUnderlining indicates SfiI restriction sites.For mycosubtilin production, strain RFB112 was cultured at 25°C for 48 h in a medium containing 1 g liter−1 yeast extract, 15 mM sucrose, 75 mM xylose, 15 mM isoleucine, 10 mM K2HPO4, 4 mM MgSO4, and 6 mM KCl. The mycosubtilin concentration in the culture broth, determined by reverse-phase high-performance liquid chromatography on a C18 X-Terra column (4.6 by 15 mm, 3.5-μm pore size; Waters) (8), was 880 mg liter−1. This represents a 50-fold-increased production yield compared to the native strain for a culture time reduced by up to 40% (8). For mycosubtilin purification, the culture supernatant was loaded onto a 50-ml C18 octadecyl silane matrix (Macherey-Nagel) equilibrated with 10 bed volumes of MilliQ water. The matrix was subsequently washed with MilliQ water and with a mixture of MilliQ water-methanol (1:1, vol/vol) (five bed volumes each). Crude mycosubtilin was then eluted with three bed volumes of methanol before being 10-fold concentrated by rotary evaporation. Further purification was performed by reverse-phase high-performance liquid chromatography, and the peak corresponding to the mycosubtilin was collected and vacuum dried. The purified molecule, analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry as previously described (13), showed signals at m/z 1,107.5 and 1,123.5 Da, which are in accordance with the calculated mass values of Na+ and K+ adducts of the C17 mycosubtilin homologue. Additional ions corresponding to C16 homologues were not detected, suggesting that the latter were not synthesized under these conditions (Fig. (Fig.1).1). Acyl chain isomery was further characterized by either 1H or 1H-13C heteronuclear single-quantum coherence NMR spectroscopy (18) on a Bruker 500-MHz spectrometer at 25°C with a 10 mM solution of purified mycosubtilin in methanol-d4. In the high-field methyl region, two signals were observed at (δH 0.89, δC 18.5) and (δH 0.93, δC 13), corresponding to a doublet and a triplet with J values of 6.9 Hz and 7.4 Hz, respectively (Fig. (Fig.2;2; data not shown). These signals, with very low proton chemical shift (δH 0.89 and δH 0.93), correspond to methyl groups of the long-chain β-amino acid found in the iturin lipopeptide (11). The proton multiplicity (i.e., doublet and triplet) observed for these two methyl groups could account only for the anteiso isomery of the heptadecanoic acyl chain of mycosubtilin.Open in a separate windowFIG. 1.Matrix-assisted laser desorption ionization-time of flight spectra of lipopeptide produced by B. subtilis RFB112. Intens., intensity; a.u., arbitrary units.Open in a separate windowFIG. 2.Detail of 1H-13C heteronuclear single-quantum coherence spectra of lipopeptides produced by B. subtilis strain RFB112. Spectra were acquired as described previously (18). The methyl signals are indicated by arrows.The biological activity of the purified anteiso-C17 mycosubtilin was first characterized by determining the susceptibility of an array of yeasts and molds. As shown in Table Table3,3, a strong antifungal activity was obtained for yeast strains whereas no significant growth inhibition was observed for molds. Despite the various susceptibilities of both yeast species and specific isolates of the same species, the relatively low MICs obtained, especially for isolates resistant to conventional drugs (fluconazole and amphotericin B), point to the powerful properties of this particular mycosubtilin isoform. The moderate effectiveness against the sterol auxotrophic Candida glabrata isolates could be explained by the presence of free ergosterol, which is known as a strong iturin antagonist (2), in the culture medium. For iturin A, an iturinic lipopeptide closely related to mycosubtilin, a membrane pore-forming mode of action has been suggested for Saccharomyces cerevisiae spheroplasts (3). Accordingly, the antifungal activity of the purified mycosubtilin was further investigated by determining its ability to induce cell membrane destabilization by measuring the transmembrane electrical potential (ΔΨ) with the fluorescent probe 3′-dipropylthia dicarbocyanine iodide [Disc(3)5] (5). As shown in Fig. Fig.3,3, the significant fluorescence signal observed upon addition of 10 μM of mycosubtilin to a Candida albicans reference strain ATCC 10231 cell suspension loaded with fluorescent dye demonstrated the complete disruption of the ΔΨ (as shown by the lack of an additional fluorescence signal upon addition of the ionophore valinomycin). At the same time, an extensive cell leakage of UV-absorbing molecules (i.e., protein and nucleic acids) (for details see reference 17) could be observed, suggesting the formation of transmembrane pores due to the action of mycosubtilin (not shown).Open in a separate windowFIG. 3.Transmembrane ΔΨ determination for a C. albicans ATCC 10231 suspension (0.5 McFarland standard). Fluorophore Disc(3)5 (1 μM; arrow 1), mycosubtilin (15 μM; arrow 2), and valinomycin (1 μM; arrow 3) were added to the cell suspension after 50, 600, and 1,000 s, respectively. Displayed data represent one representative result of three independent experiments. A.U., arbitrary units.

TABLE 3.

In vitro susceptibilities of different yeasts and molds to purified anteiso-C17 mycosubtilin
Species or strainMICa (μg/ml)Origin or referenceb
Saccharomyces cerevisiae4Lab stock
Yarrowia lipolytica CBS63038CBS
Pichia pastoris32Lab stock
Candida albicans ATCC 10231c32ATCC
Candida albicans IHEM374264IHEM
Candida albicansc18Lab stock
Candida parapsilosis IHEM9557128IHEM
Candida tropicalis IHEM624616IHEM
Candida tropicalisc,d,e16Lab stock
Candida guilliermondii IHEM1067128IHEM
Candida glabrata IHEM616116IHEM
Candida glabrata L99921
Candida glabrata S53452c,e,f1501
Candida glabrata H34736c,e,f1501
Candida glabrata W16119c,e,f1501
Candida glabratac,e16Lab stock
Cryptococcus neoformans IHEM39698IHEM
Aspergillus parasiticus IHEM4383>300IHEM
Aspergillus terreus IHEM2499>300IHEM
Aspergillus fumigatus IHEM3562>300IHEM
Open in a separate windowaResults are mean values of four independent experiments. Breakpoints for the determination of antibiotic resistance were determined according to the M27-A3 procedure (4). Experiments were performed in triplicate.bIHEM, biomedical fungus and yeast collection (http://bccm.belspo.be); ATCC, American Type Culture Collection (http://www.lgcpromochem-atcc.com); CBS, Centraalbureau voor Schimmelcultures, fungal and yeast collection (http://www.cbs.knaw.nl/databases/).cResistant to fluconazole.dResistant to amphotericin B.eClinical isolate.fAuxotroph for sterol; culture medium was supplemented with ergosterol at 20 μg/ml.Considering that the antifungal activity of iturinic lipopeptides increases with the length of their fatty acid moieties and that mycosubtilin is considered the most active iturin (10), the aim of this study was to specifically overproduce a C17 mycosubtilin homologue. As this lipopeptide is of great fundamental interest and shows great antibiotic potential, its study is particularly worthwhile. The extent to which mycosubtilin overproduction occurs in the engineered strain is greater than what has ever been observed to date (13). In combination with low culture temperature, which also contributes to this high level of production (8), addition of isoleucine made it possible to direct mycosubtilin synthesis toward the anteiso-C17 isoform with high efficiency. This preliminary investigation of the antifungal properties of this particular mycosubtilin isoform is very promising and must be pursued. We are currently investigating the mechanism of antifungal activity in greater detail, especially on Candida strains less susceptible to conventional antifungal drugs.  相似文献   

14.
Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the σW regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.Membrane-embedded proteins are crucial for cellular homeostasis and life. Membrane proteins generally account for about 30% of the open reading frames in both prokaryotic and eukaryotic genomes (49), and they are involved in a wide range of different tasks. These include vital processes, such as energy transduction, phospholipid biosynthesis, protein translocation, cell wall biogenesis, cell division, and control of cell shape (52). Importantly, membrane proteins are partially exposed to the extracytoplasmic environment, which makes them readily accessible to drugs. For this reason, membrane proteins have become a major class of proteins in terms of current drug targets. Essential membrane proteins, which are indispensable for cell proliferation under specific conditions, are especially interesting from the pharmaceutical and biomedical perspectives because they represent prime targets for chemotherapy.Unfortunately, progress in the area of membrane protein research has so far been slow. This has been attributed primarily to the high hydrophobicity of membrane proteins, which complicates high-level production, purification, and crystallization (25). Consequently, yields are often frustratingly low, as underscored by a series of elegant screens for membrane protein overproduction in Escherichia coli (10, 11, 15, 47). Moreover, the accumulation of overproduced proteins in biological membranes may affect bilayer integrity, which would be toxic for the producing cell (33). Additional limitations are potentially caused by saturation of the cellular machinery for insertion of proteins into the membrane or by saturation of the membrane itself, resulting in the cytoplasmic accumulation of overproduced membrane proteins as well as native membrane proteins (46). Such overproduced proteins are usually misfolded and/or inactive, and they have a high tendency to form insoluble (micro)aggregates. These practical problems focus attention on the fundamental question of which cellular mechanisms set the key limits to membrane protein production.In the present studies, we show that important problems in membrane protein overproduction can be overcome by using different strains of the gram-positive bacterium Bacillus subtilis as the expression host, and we identify two key mechanisms that set limits to membrane protein production in this organism. B. subtilis is highly appreciated for biotechnological applications because it has a large capacity to secrete high-quality proteins into the culture medium and because it has the status of generally recognized as safe (18, 38, 50). Furthermore, B. subtilis is amenable to genetic engineering, and many expression systems are available (2, 16, 31, 40, 43, 44). This prompted us to investigate whether the secretion machinery of B. subtilis, which is also involved in membrane protein biogenesis (52), can be exploited for membrane protein overproduction. As model proteins for our studies, we selected essential membrane proteins that have a good potential to serve as targets for novel antimicrobial drugs. Accordingly, we not only overproduced B. subtilis membrane proteins but also their orthologues from the important human pathogen Staphylococcus aureus. Studies on these essential proteins are considered to be of major relevance, since S. aureus is rapidly gaining resistance against all available antibiotics and novel antibiotics against this pathogen are urgently needed (7, 17). The results of the present studies with homologous membrane proteins from B. subtilis and S. aureus show that, like in other expression hosts, bottlenecks in membrane protein production also do exist in B. subtilis. Importantly, however, at least some of the encountered bottlenecks can be overcome, because they relate to two dispensable membrane-associated stress-responsive systems: the σW regulon and the CssRS two-component regulatory system. Thus, the removal of at least one of these stress-responsive systems can result in drastically improved yields of particular membrane proteins.  相似文献   

15.
16.
17.
18.
19.
20.
By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.  相似文献   

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