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Applied and Environmental Microbiology, May 2001, p. 2062-2069, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2062-2069.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Sequence Analysis of Insecticidal Genes from Xenorhabdus nematophilus PMFI296

J. Alun W. Morgan,1,* Martin Sergeant,1 Debbie Ellis,2 Margaret Ousley,1 and Paul Jarrett2

Department of Plant Pathology and Microbiology1 and Department of Entomological Sciences,2 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom

Received 18 October 2000/Accepted 12 February 2001

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 [LC50] of 2 to 6 µg 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 PL promoter were shown to have insecticidal activity (LC50 of 112 µg 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.

* Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom. Phone: 01789-470382. Fax: 01789-470552. E-mail: alun.morgan{at}hri.ac.uk.

Applied and Environmental Microbiology, May 2001, p. 2062-2069, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2062-2069.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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