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Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296
J A Morgan1, M Sergeant, D Ellis
1Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom. alun.morgan@hri.ac.uk
Abstract:
Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.
Insights
Xenorhabdus nematophilus bacteria exhibit insecticidal properties. Researchers identified and cloned insecticidal genes from X. nematophilus, demonstrating their activity when expressed in E. coli, paving the way for novel biopesticides.
Area of Science:
- Microbial Genetics
- Insect Pathology
- Molecular Biology
Background:
- Three strains of Xenorhabdus nematophilus possess insecticidal activity against Pieris brassicae larvae.
- Understanding the genetic basis of this insecticidal activity is crucial for developing biological control agents.
Purpose of the Study:
- To identify and characterize the genes responsible for the insecticidal activity of X. nematophilus.
- To investigate the expression and function of these genes in a heterologous host, Escherichia coli.
Main Methods:
- Construction of a cosmid genomic library from X. nematophilus PMFI296 in E. coli.
- Screening of the library for oral insecticidal activity against P. brassicae larvae.
- DNA sequencing of active cosmid clones and functional analysis using transposon mutagenesis.
Main Results:
- Two cosmid clones conferred insecticidal activity when expressed in E. coli, with LC(50) values ranging from 2 to 6 µg/g of diet.
- Sequencing revealed genes homologous to insecticidal toxins (xptA1, xptA2, xptB1, xptC1, xptD1) and a chitinase (chi).
- The xptA1 gene was essential for activity, while xptB1 and xptC1 contributed to full activity. Genes xptA2, xptD1, and chi were not critical.
Conclusions:
- A cluster of genes, including xptA1, xptB1, and xptC1, on a pathogenicity island in X. nematophilus encodes insecticidal proteins.
- Individual expression of the xptA1 gene in E. coli resulted in significant insecticidal activity, unlike previously studied toxins from Photorhabdus luminescens.
- This study provides insights into the genetic determinants of X. nematophilus insecticidal activity and its potential for biopesticide development.
