| Secretion of human interleukin-2 in biologically active form by Bacillus brevis directly into culture medium.
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Takimura Y.,Kato M.,Ohta T.,Yamagata H.,Udaka S.
Biosci. Biotechnol. Biochem. 61 (1997) 1858-61 [PMID: 9404065] Abstract
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| Abstract: We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis. |
| High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system.
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Kobayashi T.,Tamura H.,Taguchi R.,Udaka S.,Ikezawa H.
Jpn. J. Med. Sci. Biol. 49 (1996) 103-12 [PMID: 8950642] Abstract
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| Abstract: We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC. |
| Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov.
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Shida O.,Takagi H.,Kadowaki K.,Komagata K.
Int. J. Syst. Bacteriol. 46 (1996) 939-46 [PMID: 8863420] Abstract
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| Abstract: 16S rRNA gene sequences of the type strains of 11 species belonging to the Bacillus brevis and Bacillus aneurinolyticus groups were determined. On the basis of the results of gene sequence analyses, these species were separated into two clusters. The B. brevis cluster included 10 species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus formosus, Bacillus borstelensis, Bacillus laterosporus, and Bacillus thermoruber. Bacillus aneurinolyticus and Bacillus migulanus belonged to the B. aneurinolyticus cluster. Moreover, the two clusters were phylogenetically distinct from other Bacillus, Amphibacillus, Sporolactobacillus, Paenibacillus, and Alicyclobacillus species. On the basis of our data, we propose reclassification of the B. brevis cluster as Brevibacillus gen. nov. and reclassification of the B. aneurinolyticus cluster as Aneurinibacillus gen. nov. By using 16S rRNA gene sequence alignments, two specific PCR amplification primers were designed for differentiating the two new genera from each other and from other aerobic, endospore-forming organisms. |
| Bacillus brevis Migula 1900 taxonomy: reassociation and base composition of DNA.
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Nakamura LK.
Int. J. Syst. Bacteriol. 41 (1991) 510-5 [PMID: 1742197] Abstract
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| Abstract: Of 87 strains previously identified as Bacillus brevis Migula 1900, 58 had G + C contents of 47.0 to 51.9 mol%, a range that included the G + C content (48.7 mol%) of the type strain. The G + C contents for three other groups consisting of 5, 7, and 17 strains were 37.0 to 41.9, 42.0 to 46.9, and 52.0 mol% or higher, respectively. DNA reassociation studies showed that 25 of the 58 strains with G + C contents of 47.0 to 51.9 mol% were closely related genetically to the type strain and to each other. For the most part, this genetically related group was phenotypically homogeneous; variations in the fermentation of mannitol and mannose were observed. My results strongly suggest that many of the strains were misclassified as B. brevis. Consequently, much of the phenotypic heterogeneity of the species B. brevis Migula 1900 is not due to variations exhibited by genetically related organisms, but is the result of variability introduced by the presence of genetically unrelated strains. |
| Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.
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Yamagata H.,Nakahama K.,Suzuki Y.,Kakinuma A.,Tsukagoshi N.,Udaka S.
Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 3589-93 [PMID: 2786200] Abstract
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| Abstract: Using previously isolated Bacillus brevis strains that secrete large amounts of proteins but little protease into the medium, we have developed a host-vector system for very efficient synthesis and secretion of heterologous proteins. The multiple promoters and the signal-peptide-coding region of the MWP gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, were used to construct expression-secretion vectors. With this system, a synthetic gene for human epidermal growth factor (hEGF) was expressed efficiently and a large amount (0.24 g per liter of culture) of mature hEGF was secreted into the medium. hEGF purified from the culture supernatant had the same NH2-terminal amino acid sequence, COOH-terminal amino acid, and amino acid composition as natural hEGF, and it was fully active in biological assays. These results, in combination with previous results, showed that mammalian proteins can be produced in active form 10-100 times more efficiently in B. brevis than has been reported in other systems. |
| Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.
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Yamada H.,Tsukagoshi N.,Udaka S.
J. Bacteriol. 148 (1981) 322-32 [PMID: 7287624] Abstract
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| Abstract: Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers. |
| Screening for Protein-producing Bacteria.
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Udaka S.
Agric. Biol. Chem. 40 (1975) 523-528 Abstract
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| Abstract: Screening of protein-producing bacteria was conducted to systematically study the ubiquitous nature of the extracellular production of proteins and their excretion mechanisms. A very simple and efficient test revealed that about 15% of bacteria tested (total 1200 strains) accumulated some protein under the cultural conditions employed. Among protein-excretors, five strains produced a large amount of protein in the liquid shake culture.
Many good protein producers including five excellent ones were found to be gram-positive rod and probably belong to Bacillus species. An acid-insoluble product by one of the hyperprotein producers was identified as a protein mixture. Good producer was not found among the known 15 species of bacteria. The implication of these findings is discussed. |
