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 (>10(2)-10(3) 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.

Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3.

Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone-5.71, soya peptone-4.9, dextrose-1.45, NaCl-6.08, and dipotassium phosphate-0.47 in g/L). To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages.

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC50 36 hr after treatment, G. mellonella (LC50 = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC50 = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC50 = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode's symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.

Extracellular novel metalloprotease from Xenorhabdus indica and its potential as an insecticidal agent.

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

Leucobacter iarius sp. nov., in the family Microbacteriaceae.

A novel Gram-positive bacterium, strain 40(T), was isolated in the course of identifying bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum. Based on 16S rRNA gene analysis, strain 40(T) was found to be related to the type strains of recognized species of the genus Leucobacter, family Microbacteriaceae. The 16S rRNA gene sequence similarity values of strain 40(T) and Leucobacter albus IAM 14851(T), Leucobacter luti LMG 23118(T), Leucobacter alluvii LMG 23117(T), Leucobacter komagatae DSM 8803(T), Leucobacter chromiireducens CIP 108389(T) and Leucobacter aridicollis CIP 108388(T), respectively, were 97.3, 97.5, 97.6, 97.6, 97.6 and 98.5 %. Chemotaxonomic analysis also supported the affiliation of strain 40(T) to the genus Leucobacter: the major menaquinone was MK-11, the peptidoglycan cross-linkage was of the B-type, the cell wall diamino acid was L-diaminobutyric acid and the major fatty acids were anteiso-C(15 : 0) (42 %), anteiso-C(17 : 0) (34 %) and iso-C(16 : 0) (16 %). Based upon the biochemical and genomic analyses, strain 40(T) is sufficiently distinct from the type strains of recognized Leucobacter species to warrant the description of a novel species, for which the name Leucobacter iarius sp. nov. is proposed. The type strain is strain 40(T) (=DSM 17402(T)=CIP 108831(T)).

Providencia vermicola sp. nov., isolated from infective juveniles of the entomopathogenic nematode Steinernema thermophilum.

In the course of isolating bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum Ganguly & Singh, 2000, three isolates were obtained (OP1T, OP29 and VS3). On the basis of 16S rRNA gene sequence analysis and riboprint patterns, these three strains were identical to each other but distinct from the type strains of the five recognized species of the genus Providencia. Based on biochemical and genomic analysis and supported by the low (<35 %) DNA-DNA relatedness between strain OP1T and the type strain of its phylogenetically closest relative, Providencia rettgeri (99.5 % 16S rRNA gene sequence similarity), strain OP1T was considered to be sufficiently distinct from recognized Providencia species to warrant the description of a novel species. The name Providencia vermicola sp. nov. is proposed, with OP1T (= DSM 17385T = CIP 108829T) as the type strain.

A novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus indica sp. nov., symbiotically associated with entomopathogenic nematode Steinernema thermophilum Ganguly and Singh, 2000.

In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28(T) = DSM 17382(T), strain 42 = DSM 17383 and strain 43 = DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28(T) and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28(T) were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28(T), DSM 17382(T), CIP 108830(T).