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Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

ABSTRACT: A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5-10, and 14-60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.

SUBMITTER: Chang YT

PROVIDER: S-EPMC3565265 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

Chang Yu-Tzu YT Hsieh Chienyan C Wu Li-Ching LC Chang Hebron C HC Kao Suey-Sheng SS Meng Menghsiao M Hsieh Feng-Chia FC

International journal of molecular sciences 20121221 1

A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5-10, and 14-60 °C, respectively. I ...[more]

PMID: 23344035

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Project description:UnlabelledPhotorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity.ImportanceThe Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity.

Dataset Information

Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

Publications

Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

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