Characterization ofBacillus thuringiensissubsp.kurstakistrain S93 effective against the fall armywormSpodoptera frugiperda)

1999 ◽  
Vol 45 (6) ◽  
pp. 464-471 ◽  
Author(s):  
Joseilde O Silva-Werneck ◽  
Marlene T De-Souza ◽  
José MC de S. Dias ◽  
Bergmann M Ribeiro
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Fall Armyworm ◽  
Amino Acid Sequences ◽  
Dna Library ◽  
Sds Page ◽  
Total Dna ◽  
Type Gene ◽  
High Degree

A Brazilian strain of Bacillus thuringiensis subsp. kurstaki, designated S93, was analyzed regarding its cry gene and protein contents and activity against the fall armyworm (Spodoptera frugiperda, Smith 1797). Bioassays using lyophilized powders of S93 or HD-1 and third instar larvae of S. frugiperda showed a 12.3-fold lower LC50for the S93 strain when compared with the standard HD-1 strain. The spore-crystal mixture, analyzed by SDS-PAGE, showed two major polypeptides of 130 and 65 kDa, corresponding to Cry1 and Cry2 toxins, respectively. Western blot analysis showed that these proteins were immunologically related to the Cry1A protein from B. thuringiensis subsp. kurstaki HD-73. The polymerase chain reaction technique (PCR) using total DNA from the S93 strain and specific primers showed the presence of cry1Aa, cry1Ab, and cry1Ac genes, and a cry1A-type gene was localized in a plasmid of about 44 MDa. A cry1Ab gene was isolated from a S93 plasmid DNA library and completely sequenced. Computer analysis showed that the gene sequence (GenBank acession number AF059670) is identical to cry1Ab1 and has 91.6 and 85.9% identity with cry1Aa1 and cry1Ac1 genes, respectively. The deduced amino-acid sequence showed a high degree of similarity with the amino-acid sequences of the Cry1Ab1 (100%), Cry1Aa1 (93.8%), and Cry1Ac1 (90.6%) proteins.Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, crystal protein, cry genes.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

Molecular characterization and distribution of Bacillus thuringiensis cry1 genes from Brazilian strains effective against the fall armyworm, Spodoptera frugiperda

2010 ◽  
Vol 53 (3) ◽  
pp. 360-366 ◽  
Author(s):  
Fernando Hercos Valicente ◽  
Edgard Augusto de Toledo Picoli ◽  
Maria José Vilaça de Vasconcelos ◽  
Newton Portilho Carneiro ◽  
Andréia Almeida Carneiro ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Molecular Characterization ◽  
Spodoptera Frugiperda ◽  
Fall Armyworm ◽  
Cry1 Genes

scholarly journals Insecticidal activity of culture supernatants from Bacillus thuringiensis Berliner strains against Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) larvae

1999 ◽  
Vol 28 (4) ◽  
pp. 675-685 ◽  
Author(s):  
Marliton R. Barreto ◽  
Leandro L. Loguercio ◽  
Fernando H. Valicente ◽  
Edilson Paiva
Keyword(s):  
Bacillus Thuringiensis ◽  
Protein Secretion ◽  
Spodoptera Frugiperda ◽  
Wide Spectrum ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fall Armyworm ◽  
Tropical Maize ◽  
Potential Applicability ◽  
Culture Supernatants

Novel vegetative insecticidal proteins (Vips) identified in the supernatant of Bacillus thuringiensis (B.t.) cultures have shown to provide adequate control over a wide spectrum of economically important crop pests. To evaluate the potential applicability of these proteins against fall armyworm (Spodoptera frugiperda Smith) larvae, the most important insect pest for tropical maize, the characteristics and mortality effects of culture supernatants from five B.t. strains were investigated. Striking differences among strains were detected, not only in terms of efficiency in killing the insect, but also regarding to mortality effects of heated and non-heated supernatants, which were used to distinguish the heat-sensitive protein-derived insecticidal fraction from a thermostable one, with a non-protein nature (b-exotoxinas). The qualitative, quantitative and temporal patterns of total protein secretion in the medium (supernatant) were assessed through spectrophotometry and polyacrylamide gel electrophoresis. The strains showed remarkably distinct rates of growth and timing for protein secretion relative to cell density in culture. Moreover, the electrophoretic-banding patterns also varied in a strain-specific manner, both in denaturing and non denaturing conditions. Polypeptides displaying a molecular weight that is very close to the expected for previously identified Vip3A proteins were found for the strains with high supernatant-mortality ratios. The data suggest the feasibility and usefulness of searching for protein-derived (Vip-like) insecticidal fractions in B.t. supernatants as a mean of developing especific and efficient alternatives of biological control to be employed in integrated pest management programs of S. frugiperda in tropical maize.

scholarly journals Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfatigenes: purification, properties and primary sequence

2001 ◽  
Vol 357 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Karin DENGER ◽  
Jürgen RUFF ◽  
Ulrike REIN ◽  
Alasdair M. COOK
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Thiamine Pyrophosphate ◽  
Strictly Anaerobic ◽  
Laser Desorption Ionization ◽  
Native Form ◽  
Ionization Time ◽  
Sds Page

The strictly anaerobic bacterium Desulfonispora thiosulfatigenes ferments taurine via sulphoacetaldehyde, which is hydrolysed to acetate and sulphite by sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12). The lyase was expressed at high levels and a two-step, 4.5-fold purification yielded an apparently homogeneous soluble protein, which was presumably a homodimer in its native form; the molecular mass of the subunit was about 61kDa (by SDS/PAGE). The mass was determined to be 63.8kDa by matrix-assisted laser-desorption ionization–time-of-flight (MALDI–TOF) MS. The purified enzyme converted 1mol of sulphoacetaldehyde to 1mol each of sulphite and acetate, but no requirement for thiamine pyrophosphate (TPP) was detected. The N-terminal and two internal amino acid sequences were determined, which allowed us to generate PCR primers. The gene was amplified and sequenced. The DNA sequence had no significant homologue in the databases searched, whereas the derived amino acid sequence indicated an oxo-acid lyase, revealed a TPP-binding site and gave a derived molecular mass of 63.8kDa.

Carnivora: The Amino Acid Sequence of the Adult European Polecat (Mustela putorius, Mustelidae) Hemoglobins

1989 ◽  
Vol 44 (7) ◽  
pp. 817-824 ◽  
Author(s):  
Aftab Ahmed ◽  
Meeno Jahan ◽  
Gerhard Braunitzer ◽  
Helmut Pechlaner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gas Phase ◽  
Primary Structure ◽  
Amino Acid Sequences ◽  
Mustela Putorius ◽  
Globin Chains ◽  
The Family ◽  
High Degree ◽  
Β Chain

The complete amino acid sequences of the hemoglobins from the adult European polecat (Mustela putorius) are presented. The erythrocytes contain two hemoglobin components and three globin chains (α I, α II and β). The primary structure of globin chains and of the tryptic peptides determined in liquid- and gas-phase sequantors. Comparing the sequences of the globin chains of the polecat with that of human Hb-A, 17 (23.9%) substitutions were recognized in the α I, 16 (22.5%) in the α II and 14 (20.4%) in the β chain. A high degree of homology observed with other representatives of the family Mustelidae.

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp.

2005 ◽  
Vol 51 (10) ◽  
pp. 875-880 ◽  
Author(s):  
Gou Furusawa ◽  
Takeshi Yoshikawa ◽  
Yoshitaka Takano ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Band ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Pcr Product ◽  
Nutrient Agar Medium ◽  
Sds Page ◽  
Tail Sheath ◽  
A Subunit ◽  
Triton X

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200–300 nm in length. SDS–PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.Key words: Saprospira, gliding motility, Saprospira cytoplasmic fibril protein (SCFP).

scholarly journals Biorational management of maize fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) using Bacillus thuringiensis (Berliner) enriched with chemical additives

2021 ◽  
Vol 13 (4) ◽  
pp. 1231-1237
Author(s):  
M. Priyanka ◽  
P. Yasodha ◽  
C.Gailce Leo Justin ◽  
J. Ejilane ◽  
Venugopal Rajanbabu
Keyword(s):  
Bacillus Thuringiensis ◽  
Boric Acid ◽  
Sodium Nitrate ◽  
Spodoptera Frugiperda ◽  
Seedling Emergence ◽  
Fall Armyworm ◽  
Invasive Pest ◽  
Chemical Additives ◽  
Stage Of Development ◽  
Lepidoptera Noctuidae

An invasive pest, fall armyworm, Spodoptera frugiperda (J.E.Smith) (Lepidoptera: Noctuidae) attacks maize at every stage of development, from seedling emergence up to cob formation. Early instar larvae were seen mostly on leaves of maize with characteristics pin or shot hole symptoms. Later instar larvae were confined to deep whorls, leaving typically ragged like appearance and fed on the reproductive stage of the crop especially tassels and developing cobs resulting in quality and quantity loss of maize produce. The effect of commercially available Bacillus thuringiensis subsp. kurstaki product, Dipel® against the second instar larvae of Fall Armyworm (FAW )was not promising under laboratory conditions. Hence, an effort was made to add an adjuvant along with B. thuringiensis to increase the virulence of commercially available B. thuringiensis.The Laboratory bioassays with B. thuringiensis and seven chemical additives ( T1- Bt + Boric acid, T2- Bt + Zinc oxide, T3- Bt + Sodium nitrate, T4- Bt + Peptone, T5- Bt + Urea, T6- Bt + EDTA, T7- Bt + Citric acid & T8-  Bt alone T9- Control) were tested against second instar larvae of Spodoptera frugiperda larvae. The results showed that B. thuringiensis plus sodium nitrate (T3) promoted maximum mortality 82.2 per cent with a minimum LC50 value of 54.620 mg/l. Sodium nitrate boosted B. thuringiensis activity at a concentration of 0.05 per cent by 2.128-fold than B. thuringiensis alone. Overall, sodium nitrate improved the efficacy of B. thuringiensis spray at the maximum level followed by boric acid, urea, EDTA and peptone.

scholarly journals Bacillus thuringiensisCry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms,Spodoptera frugiperda(J.E. Smith)

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Yanfei Wang ◽  
Jinling Wang ◽  
Xiaoran Fu ◽  
Jeffrey R. Nageotte ◽  
Jennifer Silverman ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Protein Interactions ◽  
Spodoptera Frugiperda ◽  
Insect Cells ◽  
Fall Armyworm ◽  
Insecticidal Protein ◽  
Row Crops ◽  
Content Type ◽  
Resistance Risk ◽  
Important Pest

ABSTRACTTwo new modifiedBacillus thuringiensis(Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW),Spodoptera frugiperda(J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially availableBtproteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCEThere is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW),Spodoptera frugiperda, a maize pest that already has developed resistance toBacillus thuringiensis(Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially availableBtproteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

scholarly journals New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

2008 ◽  
Vol 74 (22) ◽  
pp. 6997-7001 ◽  
Author(s):  
Suxia Guo ◽  
Mei Liu ◽  
Donghai Peng ◽  
Sisi Ji ◽  
Pengxia Wang ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Meloidogyne Hapla ◽  
Microscopy Observation ◽  
Root Knot Nematode ◽  
Dna Library ◽  
Sds Page ◽  
New Strategy ◽  
Key Steps

ABSTRACT We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

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