Journal of Bacteriology
Công bố khoa học tiêu biểu
* Dữ liệu chỉ mang tính chất tham khảo
A mutant of
From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.
Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.
Cells of Leptothrix discophora SS1 released Mn2+-oxidizing factors into the medium during growth in batch culture. Manganese was optimally oxidized when the medium was buffered with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at pH 7.5. Manganese-oxidizing activity in the culture medium in which this strain had been grown previously was sensitive to heat, phosphate, Tris, NaN3, HgCl2 NaCl, sodium dodecyl sulfate, and pronase; 0.5 mol of O2 was consumed per mol of MnO2 formed. During Mn2+ oxidation, protons were liberated. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein-containing bands were detected in the spent culture medium. One band had an apparent molecular weight of 110,000 and was predominant in Mn2+-oxidizing activity. The second product (Mr 85,000) was only detected in some cases and probably represents a proteolytic breakdown moiety of the 110,000-Mr protein. The Mn2+-oxidizing factors were associated with the MnO2 aggregates that had been formed in spent culture medium. After solubilization of this MnO2 with ascorbate, Mn2+-oxidizing activity could be recovered.
Những sự không đồng nhất trong các gen 16S rRNA từ các chủng riêng lẻ của Paenibacillus polymyxa đã được phát hiện thông qua sự phân tách phụ thuộc vào trình tự của các sản phẩm PCR bằng phương pháp điện di gel nhiệt độ gradient (TGGE). Một đoạn của các gen 16S rRNA, bao gồm các vùng biến đổi V6 đến V8, đã được sử dụng làm trình tự mục tiêu cho các phản ứng khuếch đại. Các sản phẩm PCR từ P. polymyxa (chủng loại) đã xuất hiện như một mô hình rõ ràng với các dải trên gel gradient. Sáu plasmid với các chèn khác nhau, thể hiện các đặc điểm di chuyển của các dải đơn lẻ trong mô hình, đã được thu được thông qua việc nhân bản các sản phẩm PCR. Các chuỗi của chúng đã được phân tích như một mẫu đại diện cho sự không đồng nhất tổng thể. Một số 10 vị trí nucleotide biến thể trong đoạn dài 347 bp đã được quan sát, với tất cả các sự thay thế vẫn bảo tồn cấu trúc thứ cấp liên quan của các vùng V6 và V8 trong các phân tử RNA. Sự lai ghép với các mồi được thiết kế đặc biệt đã chứng minh các vị trí nhiễm sắc thể khác nhau của các gen rRNA tương ứng. Các phản ứng khuếch đại của rRNA chuyển đảo từ các chuẩn bị ribosome, cũng như lai ghép toàn tế bào cho thấy một sự đại diện vượt trội của các trình tự cụ thể trong ribosome của các văn hóa phòng thí nghiệm đang tăng trưởng theo cấp số nhân. Các chủng khác nhau của P. polymyxa không chỉ cho thấy các mô hình rất khác nhau của sản phẩm PCR trong phân tích TGGE mà còn có sự gán nhãn toàn tế bào phân biệt với các mồi oligonucleotide đã thiết kế, cho thấy sự đại diện khác nhau của các trình tự riêng trong các ribosome hoạt động. Các kết quả của chúng tôi chứng minh tính hữu ích của TGGE trong phân tích cấu trúc của các gen rRNA không đồng nhất cùng với biểu hiện của chúng, nhấn mạnh các vấn đề về việc tạo ra dữ liệu có ý nghĩa cho các trình tự 16S rRNA và thiết kế mồi, và có thể có những hệ quả đối với các khái niệm tiến hóa.
There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.
The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.
A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.
The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes. A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT. The nucleotide sequence of bglP showed a high degree of homology with the E. coli bglF gene (BglF is a beta-glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator). Complementation studies of an E. coli strain with a deletion of the bgl operon showed that BglP was a functional beta-glucoside permease. In B. subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion. Disruption of licT abolished the suppressor phenotype in a bglP mutant. LicT is a recently identified third B. subtilis antiterminator of the BglG/SacY family. These observations indicated that BglP was also a negative regulator of LicT. Both LicT and BglP seem to be involved in the induction by beta-glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho-beta-glucosidase. Other beta-glucoside genes homologous to bglP and bglH have been recently described in B. subtilis. Thus, B. subtilis possesses several sets of beta-glucoside genes, like E. coli, but these genes do not appear to be cryptic.
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