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Identification of residues in domain III of Bacillus thuringiensis Cry1Ac toxin that affect binding and toxicity

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第10期65卷 4513-4520页

作者： Lee, MK You, TH Gould, FL Dean, DH Ohio State Univ Dept Biochem Columbus OH 43210 USA N Carolina State Univ Dept Entomol Raleigh NC 27695 USA

Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues (SS504)-S-503, (NNI508)-N-506, (509)QNR(511), (ST523)-S-522, and (ST525)-S-524. Single alanine substitutions were made at the residues (509)Q, N-510, R-511, and Y-513. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations, (50)3SS(504)-AA, (NNI508)-N-506-AAA, (ST523)-S-522-AA, (ST525)-S-524-AA, and N-510-A affected neither the protein's toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the (509)QNR(511)-AAA, (509)Q-A, R-511-A, and Y-513-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the (509)QNR(511)-AAA, R-511-A, R-511-A, and Y-513-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only (509)QNR(511)-AAA and R-511-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues (509)Q, R-511, and Y-513 in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.

关键词：细菌蛋白质类/化学细菌蛋白质类/代谢细菌蛋白质类/药理学细菌毒素类内毒素类/化学内毒素类/代谢内毒素类/药理学溶血素蛋白质类杀虫药/化学天蛾微绒毛/代谢诱变定点构效关系动物

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Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens

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JOURNAL OF FOOD PROTECTION 1999年第9期62卷 1011-1016页

作者： Morgan, SM Galvin, M Kelly, J Ross, RP Hill, C TEAGASC Dairy Prod Res Ctr Fermoy Cork Ireland Univ Coll Natl Food Biotechnol Ctr Cork Ireland Univ Coll Dept Microbiol Cork Ireland

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10(8) midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C. In the case of S. aureus 10, resuspension of 2.5 X 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30 degrees C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.

关键词：细菌蛋白质类/药理学细菌素类/药理学培养基食品微生物学革兰氏阳性菌/药物作用革兰氏阳性菌/致病力氢离子浓度单核细胞增生利斯特菌/药物作用单核细胞增生利斯特菌/致病力乳/微生物学乳蛋白质类/化学金黄色葡萄球菌/药物作用金黄色葡萄球菌/致病力动物

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MECHANISM OF ACTION OF SALMONELLA PHAGE-P22 ANTIREPRESSOR

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JOURNAL OF MOLECULAR BIOLOGY 1975年第2期98卷 413-424页

作者： SUSSKIND, MM BOTSTEIN, D MIT DEPT BIOLCAMBRIDGEMA 02139

On the basis of genetic evidence, Botstein et al. (1975) and Levine et al. (1975) proposed that phage P22 specifies an antirepressor which interferes with the synthesis or activity of the P22 c 2 repressor. In this paper, we show that P22 anti-repressor interferes with phage λ cI repressor (which is analogous in function to P22 c 2 repressor), since superinfection of a λ lysogen with P22 results in dis-appearance of λ repressor activity. Antirepressor interferes directly with repressor activity rather than indirectly with repressor synthesis, since antirepressor in crude P22-infected cell extracts inhibits λ repressor activity in vitro . Under our assay conditions, antirepressor action requires pre-incubation of antirepressor with λ repressor before addition of λ DNA, suggesting that antirepressor works by interacting with repressor rather than by interacting with the DNA operators. We can rule out the hypothesis that antirepressor inactivates repressor by covalently modifying or cleaving it, since repressor that has been inactivated by antirepressor can be recovered in active form by treatment with sodium dodecyl sulfate and urea, or by treatment with guanidine. Taken together, these results suggest that antirepressor inactivates repressor by binding non-covalently to it.

关键词：等位基因细菌蛋白质类/药理学动力学溶源性突变沙门菌噬菌体/药物作用沙门菌噬菌体/代谢鼠伤寒沙门菌/代谢种特异性病毒复制/药物作用

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Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第3期88卷 414-420页

作者： Morgan, SM Ross, RP Beresford, T Hill, C TEAGASC Dairy Prod Res Ctr Fermoy Cork Ireland Natl Univ Ireland Univ Coll Cork Natl Food Biotechnol Ctr Cork Ireland Natl Univ Ireland Univ Coll Cork Dept Microbiol Cork Ireland

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.

关键词：细菌蛋白质类/药理学细菌素类/药理学集落计数微生物食品保存/方法流体静力压利斯特菌属/药物作用利斯特菌属/生长和发育乳/微生物学乳蛋白质类金黄色葡萄球菌/药物作用金黄色葡萄球菌/生长和发育动物

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Interactions of a novel inhibitor from an extremophilic Bacillus sp with HIV-1 protease -: Implications for the mechanism of inactivation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第4期276卷 2487-2493页

作者： Dash, C Rao, M Natl Chem Lab Div Biochem Sci Pune 411008 Maharashtra India

The active site cleft of the HIV-1 protease (PR) is bound by two identical conformationally mobile loops known as flaps, which are important for substrate binding and catalysis, The present article reports, for the first time, an HIV-1 PR inhibitor, ATBI, from an extremophilic Bacillus sp. The inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu, Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1 PR. Investigation of the kinetics of the enzyme-inhibitor interactions revealed that ATBI is a noncompetitive and tight binding inhibitor with the IC50 and K-i values 18.0 and 17.8 nM, respectively. The binding of the inhibitor with the enzyme and the subsequent induction of the localized conformational changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps. We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the active site of the HIV-1 PR and thereby leads to the inactivation of the enzyme. Based on our results, we propose that the inactivation is due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of the enzyme.

关键词：氨基酸序列芽孢杆菌属细菌蛋白质类/化学细菌蛋白质类/药理学 HIV蛋白酶/药物作用 HIV蛋白酶抑制药/化学 HIV蛋白酶抑制药/药理学 HIV-1/酶学质谱分析法模型生物学模型分子寡肽类/化学寡肽类/药理学蛋白质结合蛋白质构象/药物作用蛋白质结构二级序列分析蛋白质

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Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by activating NF-κB via Ref-1

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第2期276卷 1335-1344页

作者： Daily, D Vlamis-Gardikas, A Offen, D Mittelman, L Melamed, E Holmgren, A Barzilai, A Tel Aviv Univ George S Wise Fac Life Sci Dept Neurobiochem IL-69978 Tel Aviv Israel Karolinska Inst Med Nobel Inst Biochem Dept Biochem & Biophys S-17177 Stockholm Sweden Tel Aviv Univ Rabin Med Ctr Dept Neurol IL-69978 Tel Aviv Israel Tel Aviv Univ Rabin Med Ctr Felsenstein Med Res Inst IL-69978 Tel Aviv Israel Tel Aviv Univ Sackler Sch Med Interdept Core Facil IL-69978 Tel Aviv Israel

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis, E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappaB activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-kappaB binding activity, Grx2 could stimulate the binding of NF-kappaB to DNA by: (i) translocating NF-kappaB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappaB alpha, and (ii) activating the binding of pre existing nuclear NF-kappaB. The DNA binding activity of NF-kappaB itself was essential for neuronal survival. Overexpression of I-kappaB dominant negative gene (I-kappaB-DeltaN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappaB binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappaB.

关键词：氨基酸取代动物新生细胞凋亡/药物作用细胞凋亡/生理学细菌蛋白质类/药理学碳氧裂合酶类/代谢细胞培养的小脑/细胞学 DNA修复 DNA-(无嘌呤或无嘧啶位点)裂合酶多巴胺/药理学内切脱氧核糖核酸酶类/代谢大肠杆菌小鼠近交BALBC 诱变定点 NF-κB/代谢神经元/细胞学神经元/药物作用神经元/生理学氧化还原酶类蛋白质转运/药物作用蛋白质类/化学蛋白质类/遗传学蛋白质类/药理学重组蛋白质类/药理学动物人类小鼠

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Hyper-production of insecticidal crystal protein (δ-endotoxin) by Bacillus thuringiensis var. israelensis is not related to sporulation-specific biochemical functions

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CURRENT MICROBIOLOGY 2000年第3期41卷 187-191页

作者： Bhattacharya, PR Malaria Res Ctr Delhi 110054 India

Hypertoxic mutant strains of Bacillus thuringiensis var, israelensis were isolated by mutagenesis of the parent strain. The correlation, if any, between hyper-production of insecticidal crystal protein (delta-endotoxin) by hypertoxic mutant strains of Bacillus thuringiensis var. israelensis and sporulation-specific biochemical functions was studied. No increase in sporulation-specific biochemical markers was observed in the hypertoxic mutant strains. Asporogenous mutants of hypertoxic mutant strains blocked at different stages of sporulation were isolated, and larvicidal activity was studied. The hypertoxic parent strains and the sporulation-deficient, hypertoxic mutant strains showed almost identical larvicidal activity. Therefore, the increased production of toxin is not related to sporulation-specific biochemical changes.

关键词：芽孢杆菌苏云金/药物作用芽孢杆菌苏云金/遗传学芽孢杆菌苏云金/代谢细菌蛋白质类/代谢细菌蛋白质类/药理学细菌毒素类/代谢细菌毒素类/药理学内毒素类/代谢内毒素类/药理学甲磺酸乙酯/药理学溶血素蛋白质类幼虫/药物作用诱变孢子

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Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第45期274卷 32023-32030页

作者： Horn, C Namane, A Pescher, P Rivière, M Romain, F Puzo, G Bârzu, O Marchal, G Inst Pasteur Unite Physiopathol Infect F-75724 Paris 15 France Inst Pasteur Lab Chim Struct Macromol F-75724 Paris 15 France CNRS Inst Pharmacol & Biol Struct F-31077 Toulouse 15 France

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens, Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from II M.. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG, The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro, Thus the observed biological effects were related to the extent of glycosylation of the antigen.

关键词：氨基酸序列抗原细菌/遗传学抗原细菌/药理学细菌蛋白质类/遗传学细菌蛋白质类/药理学糖蛋白类/遗传学糖蛋白类/药理学糖基化超敏反应迟发型淋巴细胞活化/药物作用甘露糖/代谢分子序列数据分子量结核分枝杆菌重组蛋白质类/药理学光谱法质量基质辅助激光解吸电离 T淋巴细胞/药物作用 T淋巴细胞/免疫学动物豚鼠

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Identification of Bacillus thuringiensis delta-endotoxin Cry1C domain III amino acid residues involved in insect specificity

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第10期65卷 4369-4374页

作者： de Maagd, RA Bakker, P Staykov, N Dukiandjiev, S Stiekema, W Bosch, D DLO Ctr Plant Breeding & Reprod Res Dept Mol Biol NL-6700 AA Wageningen Netherlands Paisij Hilendarski Univ Plovdiv Dept Mol Biol & Plant Physiol BG-4000 Plovdiv Bulgaria

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.

关键词：氨基酸序列细菌蛋白质类/化学细菌蛋白质类/药理学细菌毒素类内毒素类/化学内毒素类/药理学溶血素蛋白质类杀虫药/化学天蛾分子序列数据诱变害虫防治生物学斜纹夜蛾属构效关系动物

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The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第41期274卷 29289-29293页

作者： Zumbihl, R Aepfelbacher, M Andor, A Jacobi, CA Ruckdeschel, H Rouot, B Heesemann, J Max Von Pettenkofer Inst Hyg & Med Microbiol D-80336 Munich Germany Univ Montpellier 2 INSERM U431 F-34095 Montpellier France

Pathogenic Yersinia enterocolitica produces two virulence plasmid-encoded cytotoxins, YopE and YopT, that are translocated into target cells where they disrupt the actin cytoskeleton. Here we show that infection of cells with wild type Y. enterocolitica and a yopE mutant, but not with a yopT mutant, induces an increase in the electrophoretic mobility of the small. GTPase RhoA As tested by isoelectric focusing, YopT-dependent modification resulted in an acidic shift of RhoA. Furthermore, RhoA modification induced by YopT was accompanied by redistribution of membrane-bound RhoA toward the cytosol, Finally, a yopE mutant of Y, enterocolitica expressing the cytotoxic activity of YopT specifically disrupted RhoA-controlled actin stress fibers. These findings provide evidence for inactivation of RhoA by the translocated Y. enterocolitica cytotoxin YopT and suggest a novel inhibitory modification of RhoA by a bacterial virulence factor.

关键词：细菌外膜蛋白质类/药理学细菌蛋白质类/药理学 COS细胞半胱氨酸内肽酶类细胞毒素类/药理学内皮血管/细胞学内皮血管/微生物学等电聚焦显微镜检查荧光突变毒力小肠结肠炎耶尔森菌/化学小肠结肠炎耶尔森菌/致病力 ρAGTP结合蛋白质/化学动物

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