Vishal Singh Somvanshi

Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill

A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain (B. subtilis), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli. The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.

Identification of virulent entomopathogenic nematode isolates from a countrywide survey in India

Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biological management of insect pests, for example, invasive mole crickets, white grubs and other scarab larvae in lawns and turf, fungus gnats in mushroom, black vine weevil and Diaprepes root weevil in nursery plants and citrus, respectively. The use of EPNs in India is still in the preliminary stages. To find the best EPN isolates for use in local insect pest management programs, we previously collected 35 strains of EPNs from different parts of India, and screened them for insect infectivity at broad temperature range. Here we quantitated the virulence of seven EPN strains showing infectivity at broad temperature range against Galleria mellonella and Spodoptera litura. Three isolates of Steinernema and four isolates of Heterorhabditis were tested. The Heterorhabditis sp. Hmg3 was the most virulent EPN isolate among all the tested nematodes (LC50 = 16.1 infective juveniles (IJs)/insect larvae at 24 h), whereas the isolate Steinernema thermophilum Kr was the most virulent steinernematid (LC50 = 11.8 IJ/insect larvae at 36 h). These strains would be taken up for mass multiplication to facilitate their actual use in insect pest management programs at farmers fields. Our results suggest that G. mellonella could be used as a primary model for assessing and predicting the virulence of EPN isolates on other insects such as S. litura.

Photorhabdus : A Microbial Factory of Insect-Killing Toxins

The overuse of chemical pesticides to meet the production and productivity goals in modern agriculture is causing a number of unintended side effects and destruction of the environment. Eco-friendly pest management techniques and strategies are urgently needed. Photorhabdus spp. are Gram-negative gamma-proteobacteria of the family Enterobacteriaceae, found exclusively in symbiotic association with nematodes of the genus Heterorhabditis. Heterorhabditis nematodes are widely used as a biological control agent for insect-pests of crops. These nematodes carry the symbiont bacteria in their gut and release them in insect hemocoel upon infection of new insect host. Inside the insect hemocoel, Photorhabdus multiplies and releases a multitude of insecticidal toxins and secondary metabolites resulting in death of the insect by septicemia and toxemia. Some of these toxins are highly specific to their target species, while others are generalists. Stand-alone formulation of Photorhabdus bacteria is reported to be selling well in markets for insect management. Photorhabdus toxins are considered next to Bt toxins in their potential for use in insect-pest management in agriculture.

Proteomic Investigation of Photorhabdus Bacteria for Nematode-Host Specificity.

Majority of animals form symbiotic relationships with bacteria. Based on the number of bacterial species associating with an animal, these symbiotic associations can be mono-specific, relatively simple (2–25 bacterial species/animal) or highly complex (>102–103 bacterial species/animal). Photorhabdus (family-Enterobacteriaceae) forms a mono-specific symbiotic relationship with the entomopathogenic nematode Heterorhabditis. This system provides a tractable genetic model for animal-microbe symbiosis studies. Here, we investigated the bacterial factors that may be responsible for governing host specificity between nematode and their symbiont bacteria using proteomics approach. Total protein profiles of P. luminescens ssp. laumondii (host nematode- H. bacteriophora) and P. luminescens ssp. akhurstii (host nematode- H. indica) were compared using 2-D gel electrophoresis, followed by identification of differentially expressed proteins by MALDI-TOF MS. Thirty-nine unique protein spots were identified - 24 from P. luminescens ssp. laumondii and 15 from P. luminescens ssp. akhurstii. These included proteins that might be involved in determining host specificity directly (for e.g. pilin FimA, outer membrane protein A), indirectly through effect on bacterial secondary metabolism (for e.g. malate dehydrogenase Mdh, Pyruvate formate-lyase PflA, flavo protein WrbA), or in a yet unknown manner (for e.g. hypothetical proteins, transcription regulators). Further functional validation is needed to establish the role of these bacterial proteins in nematode-host specificity.

A 37 kDa Txp40 protein characterized from Photorhabdus luminescens sub sp. akhurstii conferred injectable and oral toxicity to greater wax moth, Galleria mellonella

Photorhabdus luminescens is a gram-negative bacterium that symbiotically associates with insect-parasitic nematode, Heterorhabditis indica. Herein, we have characterized an insecticidal gene, Txp40 (1008 bp) from the indigenous isolates of P. luminescens, and tested its bioefficacy against Galleria mellonella via injectable and oral bioassay. The recombinant protein characterized from P. luminescens strain H3 exhibited comparatively greater insect toxicity than strain H1 in terms of LD50 and LT50 values. Txp40 holds great potential to replace Bt toxins in global agriculture.

Expression of Heterorhabditis bacteriophora C-type lectins, Hb-clec-1 and Hb-clec-78, in context of symbiosis with Photorhabdus bacteria

Insect-parasitic nematodes of the genus Heterorhabditis live in a symbiotic relationship with a gram negative Gamma-proteobacteria of the genus Photorhabdus. This nematode-bacteria pair is a simple and genetically tractable model to study animal-microbe symbiosis. Here we investigated the role of Heterorhabditis nematode C-type lectin (clec) genes in context of nematode-bacteria symbiosis. The in silico analysis identified seven clec genes in H. bacteriophora and three clec genes in H. indica. Two of the clec genes, H. bacteriophora clec-1 (Hb-clec-1) and H. bacteriophora clec-78 (Hb-clec-78) were further characterized. Both of these genes were present in a single copy in the H. bacteriophora genome. The phylogenetic analysis revealed that H. bacteriophora CLEC proteins were close to CLEC-1 and CLEC-78 proteins of free living Caenorhabditis but not to the CLEC proteins of insect-parasitic Steinernema nematodes which share a similar symbiotic relationship with Xenorhabdus bacteria. In situ hybridization showed that expression of Hb-clec-1 and Hb-clec-78 was localized to the alimentary canal of infective juveniles (IJs) in the region of terminal bulb, oesophago-intestinal valve and anterior part of intestine. Hb-clec-78 gene expression displayed significant positive correlation to the presence of bacteria during various stages of symbiosis: it was up-regulated during all the nematode developmental stages when Photorhabdus was symbiotically associated, but down-regulated at the post-IJ recovery stage when the developing nematodes were free of bacteria. Hb-clec-1 gene expression did not show any correlation with presence or absence of symbiont bacteria. Subject to genetic validation, our study suggests that Hb-clec-78 might be actively involved in modulation of symbiosis with Photorhabdus symbionts.

Silencing of a Meloidogyne incognita selenium-binding protein alters the cuticular adhesion of Pasteuria penetrans endospores

Pasteuria penetrans is an endospore forming hyperparasitic bacterium of the plant-pathogenic root-knot nematode, Meloidogyne incognita. For successful parasitization, the first step is adherence of bacterial endospores onto the cuticle surface of nematode juveniles. The knowledge of molecular intricacies involved during this adherence is sparse. Here, we identified a M. incognita selenium-binding protein (Mi-SeBP-1) differentially expressed during the initial interaction of M. incognita and P. penetrans, and show that it is involved in modulating parasitic adhesion of bacterial endospores onto nematode cuticle. Selenium-binding proteins (SeBPs) are selenium associated proteins important for growth regulation, tumor prevention and modulation of oxidation/reduction in cells. Although reported to be present in several nematodes, the function of SeBPs is not known in Phylum Nematoda. In situ hybridization assay localized the Mi-SeBP-1 mRNA to the hypodermal cells. RNAi-mediated silencing of Mi-SeBP-1 significantly increased the adherence of P. penetrans endospores to the nematode juvenile cuticle. Silencing of Mi-SeBP-1 did not change the nematodes ability to parasitize plants and reproduction potential within the host. These results suggest that M. incognita Mi-SeBP-1 might be involved in altering the attachment of microbial pathogens on the nematode cuticle, but is not involved in nematode-host plant interaction. This is the first report for a function of SeBP in Phylum Nematoda.

A comprehensive annotation for the root-knot nematode Meloidogyne incognita proteome data

Root-knot nematodes are devastating pathogens of crop plants. The draft genome of southern root-knot nematode Meloidogyne incognita was published in 2008 and additional genome and transcriptome data became available later on. However, lack of a publically available annotation for M. incognita genome and transcriptome(s) limits the use of this data for functional and comparative genomics by the interested researchers. Here we present a comprehensive annotation for the M. incognita proteome data available at INRA Meloidogyne Genomic Resources page (https://meloidogyne.inra.fr/Downloads/Meloidogyne-incognita-V2-2017) and European Nucleotide Archive (ENA) (accession number: ERP009887) using a multi-pronged approach.

Discovery of a Highly Virulent Strain of Photorhabdus luminescens ssp. akhurstii from Meghalaya, India

Photorhabdus is an insect-pathogenic Gram negative enterobacterium found in the gut of Heterorhabditis nematodes. Photorhabdus is highly virulent to insects, and can kill insects rapidly upon injection at very low concentrations of one to few cells. We characterized the virulence of Photorhabdus symbionts isolated from the Heterorhabditis nematodes collected from various parts of India by injecting different concentrations of bacterial cells into fourth instar larval stage of insect Galleria mellonella. Photorhabdus luminescens ssp. akhurstii strain IARI-SGMG3 from Meghalaya was identified as the most virulent of all the tested strains on the basis of LT50 and LC50 values. This study forms a basis for further investigations on the genetic basis of virulence in Photorhabdus bacteria.