Naresh Arora

Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis

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.

A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.

A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.

Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin

The bacterium Bacillus thuringiensis produces ICPs (insecticidal crystal proteins) that are deposited in their spore mother cells. When susceptible lepidopteran larvae ingest these spore mother cells, the ICPs get solubilized in the alkaline gut environment. Of approx. 140 insecticidal proteins described thus far, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation as well as the principal ICP introduced into crops as transgene for agricultural crop protection. The 135 kDa Cry1Ac protein, upon ingestion by the insect, is processed successively at the N- and C-terminus by the insect midgut proteases to generate a 65 kDa bioactive core protein. The activated core protein interacts with specific receptors located at the midgut epithilium resulting in the lysis of cells and eventual death of the larvae. A laboratory-reared population of Helicoverpa armigera displayed 72-fold resistance to the B. thuringiensis insecticidal protein Cry1Ac. A careful zymogram analysis of Cry1Ac-resistant insects revealed an altered proteolytic profile. The altered protease profile resulted in improper processing of the insecticidal protein and as a consequence increased the LC50 concentrations of Cry1Ac. The 135 kDa protoxin-susceptible insect larval population processed the protein to the biologically active 65 kDa core protein, while the resistant insect larval population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides. N-terminal sequencing of these 95 and 68 kDa polypeptides produced by gut juices of resistant insects revealed an intact N-terminus. Protease gene transcription profiling by semi-quantitative RT (reverse transcription)-PCR led to the identification of a down-regulated HaSP2 (H. armigera serine protease 2) in the Cry1Ac-resistant population. Protease HaSP2 was cloned, expressed and demonstrated to be responsible for proper processing of insecticidal protoxin. The larval population displaying resistance to Cry1Ac do not show an altered sensitivity against another insecticidal protein, Cry2Ab. The implications of these observations in the context of the possibility of development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation are discussed.

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquitos vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis

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.

Expression, purification, and characterization of pro-phenoloxidase-activating serine protease from Spodoptera litura.

One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6 h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni-NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N-terminal sequencing of processed phenol oxidase revealed 11 kDa propeptide instead of in-silico predicted 6 kDa polypeptide.

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.

Evidence for the shikimate‐3‐phosphate interacting site in the N‐terminal domain of 5‐enolpyruvyl shikimate‐3‐phosphate synthase of Bacillus subtilis

The role of basic amino acid residues that are highly conserved in the N‐terminal domain of 5‐enolpyruvyl shikimate‐3‐phosphate synthase (EPSPs) in the binding of the substrate, shikimate‐3‐phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site‐directed mutagenesis. Native and the mutant proteins were expressed using a two‐vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N‐terminal domain (K19 and R24) results in reduced affinity for shikimate‐3‐phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.

Biology And Applications Of Bacillus Thuringiensis In Integrated Pest anagement

The application of Bacillus thuringiensis is revised, with description of its biology, ecology and potentials for insects management. Co-evolution of the crystal proteins structure with insect hosts is reviewed, with data on the classification and nomenclature. The function of various Cry proteins and their role in insect parasitism are described together with the mechanism of action of the toxins. Applications for control of mosquitoes and blackflies and formulations are reviewed. Factors related to the development of resistance and potentials in the integrated pest management are discussed.