Angamuthu Selvapandiyan
International Centre for Genetic Engineering and Biotechnology
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Featured researches published by Angamuthu Selvapandiyan.
Applied and Environmental Microbiology | 2001
Angamuthu Selvapandiyan; Naresh Arora; Raman Rajagopal; S. K. Jalali; T. Venkatesan; S. P. Singh; Raj K. Bhatnagar
ABSTRACT A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.
Biochemical Journal | 2003
Raman Rajagopal; Neema Agrawal; Angamuthu Selvapandiyan; S. Sivakumar; Suhail Ahmad; Raj K. Bhatnagar
Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.
Molecular Breeding | 2002
Vanga Siva Reddy; Sadhu Leelavathi; Angamuthu Selvapandiyan; Rajagopal Raman; Ferraiolo Giovanni; Vijaya Shukla; Raj K. Bhatnagar
Stable inheritance and sustained-high level expression of foreign genes in the progeny are the most critical factors for successful application of genetic engineering in agriculture. In this study, we have transformed cry1Ia5 into tobacco chloroplasts and studied the expression, inheritance and resistance offered against Helicoverpa armigera over two generations. Under rice chloroplast transcription elements, the Cry1Ia5 protein accumulated up to 3% of total soluble protein in leaf tissue which is ∼ 300 folds more when compared to the expression of the same protein in the nuclear transformed plants. Transgenic plants offered complete protection against larvae of H. armigera, irrespective of development stage. Analysis of T0, T1 and T2 generation plants revealed site-specific integration, maternal inheritance and uniform expression of transgenes without imposing any yield penalty. Our results suggest that the overexpression of insecticidal toxin coding genes in chloroplasts would be an effective strategy to delay the emergence of resistance among phytophagous pests.
International Journal of Systematic and Evolutionary Microbiology | 1999
Suhail Ahmad; Angamuthu Selvapandiyan; Raj K. Bhatnagar
The dnaK operon from Bacillus subtilis and other Gram-positive bacteria with low G + C DNA content contains additional heat-shock genes, including hrcA. The hrcA gene encodes a transcription factor that negatively regulates heat-shock genes and is uniformly present in all Gram-positive bacteria studied to date. An hrcA homologue is also present in Synechocystis species, Leptospira interrogans, Chlamydia trachomatis, Caulobacter crescentus and Methanococcus jannaschii, organisms that diverged early on from the common ancestor of all Gram-positive bacteria and Proteobacteria, according to 16S rRNA phylogeny. A partial, protein-based phylogenetic tree, derived using amino acid sequence homology of hrcA proteins from Gram-positive bacteria, is presented here, and the results are compared with the phylogenetic trees generated from 16S rRNA, dnaK and dnaJ sequences. The location of the hrcA gene and the genome organization of the dnaK operon support the division of all Gram-positive bacteria into three major groups: one group contains high-G + C Gram-positive bacteria, and two others contain low-G + C Gram-positive bacteria. Among the Gram-positive bacteria with low G + C DNA content, the results indicate that there is a close phylogenetic relationship between Bacillus species and Clostridium species on the one hand and between Lactococcus lactis and Streptococcus mutans on the other. Streptomyces and Mycobacterium species also exhibited a close relationship. A hierarchical arrangement of Gram-positive bacteria based on HrcA sequences is proposed as an additional refinement of the phylogenetic relationships within this important bacterial group.
FEBS Letters | 1997
Roberto Marzari; Paolo Edomi; Raj K. Bhatnagar; Suhail Ahmad; Angamuthu Selvapandiyan; Andrew Bradbury
The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.
Molecular Breeding | 1998
Angamuthu Selvapandiyan; Vanga Siva Reddy; P. Anand Kumar; Krishna K. Tewari; Raj K. Bhatnagar
A cry1Ia5 insecticidal toxin coding gene has been cloned from an Indian isolate of Bacillus thuringiensis. Sequence analyses of the cry1Ia5 gene revealed the absence of potential polyadenylation signal sequences thus making it a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cry1Ia5 gene into a plant expression vector and then transferring it to Nicotiana tabacum through Agrobacterium-mediated transformation. Our results demonstrate that N. tabacum with a stably integrated native cry1Ia5 gene afforded complete protection against predation by Heliothis armigera. Forty three percent of the transgenic plants displayed a high level of protection against insect predation. The protection obtained in transgenic plants with the cry1Ia5 gene was comparable to that obtained with the synthetically modified cry1A(b) or cry1A(c) genes. The results demonstrate that novel insecticidal genes already exist in nature that do not require extensive modifications for efficient expression in plants.
International Journal of Systematic and Evolutionary Microbiology | 2000
Suhail Ahmad; Angamuthu Selvapandiyan; Raj K. Bhatnagar
The dnaK operon in gram-positive bacteria includes grpE, dnaJ and, in some members, hrcA as well. Both DnaK and DnaJ have been utilized for constructing phylogenetic relationships among various organisms. Multiple copies exist for dnaK and dnaJ genes in some bacterial genera, as opposed to a single gene copy for grpE and for hrcA, according to the currently available data. Here, we present a partial protein-based phylogenetic tree for gram-positive bacteria, derived by using the amino acid sequence identity of GrpE; the results are compared with the phylogenetic trees generated from 5S rRNA, 16S rRNA, dnaK and dnaJ sequences. Our results indicate three main groupings: two are within low-G+C DNA gram-positive bacteria comprising Bacillus species and Staphylococcus aureus on the one hand and Streptococcus species/Lactococcus lactis/Enterococcus faecalis/Lactobacillus sakei on the other hand; the Mycobacterium species and Streptomyces coelicolor, belonging to the high-G+C DNA gram-positive bacteria, form the third cluster. This hierarchical arrangement is in close agreement with that obtained with 16S rRNA and DnaK sequences but not DnaJ-based phylogeny.
FEBS Letters | 1995
Angamuthu Selvapandiyan; Kumud Majumder; Farkad A. Fattah; Suhail Ahmad; Naresh Arora; Raj K. Bhatnagar
The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5‐enolpyruvyl shikimate‐3‐phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate—inhibitor binding mode and constants. Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.
Applied Microbiology and Biotechnology | 1994
Angamuthu Selvapandiyan; Raj K. Bhatnagar
A Pseudomonas isolate (GLC11) capable of growth in the presence of up to 125 mM glyphosate [N-phosphonomethyl glycine (PMG)] has been isolated. Unlike the previously isolated Pseudomonas PG2982 and other bacterial strains, isolate GLC11 grows equally well in commercial formulation and analytical grade PMG. Utilisation of PMG as a phosphorus source is repressed by inorganic phosphate (Pi) in both isolates. Enzymatic activity responsible for carbon-phosphorus bond cleavage (C-P lyase) was detected in cell-free extracts of both isolates and was partially purified. Resolution on DE-52 anion exchange chromatography yielded a single peak of C-P lyase activity. The molecular mass of C-P lyase as analysed by gel permeation chromatography is approximately 200 kDa. The enzyme activity was localised in the periplasmic space of bacteria. The specific activity of C-P lyase was different for different phosphonates when used as substrates.
Applied Microbiology and Biotechnology | 1998
Suhail Ahmad; Angamuthu Selvapandiyan; Raj K. Bhatnagar
Abstract The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system. The expressed proteins are readily visible in Coomassie-blue-stained protein gels. The recombinant E. coli cells expressing toxic proteins were toxic towards Culex larvae. During the assembly of crystals in B. sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B. sphaericus protease. To express the toxins in E. coli the B.sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal. This modification resulted in a twofold increase in the toxicity of the E. coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin. Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B. sphaericus.
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International Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
View shared research outputsInternational Centre for Genetic Engineering and Biotechnology
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