Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

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.

FIG. 1

SDS-PAGE analysis of cell extracts from X. nematophilus pMFI296 and E. coli clones. (A) Lane 1, X. nematophilus pMFI296; lane 2, E. coli; lane 3, E. coli (cHRIM1); and lane 4, E. coli (338/2) stained with coomassie brilliant blue. (B) Lane 1, E. coli (cHRIM1) stained with SYPRO Orange. The position of XptA1 is marked with an arrow. Size markers (M) are presented (in kilodaltons).

FIG. 2

Characterization of the contribution of genes on cHRIM1 to insecticidal activity. (A) Map indicating the effect of transposon insertions in genes, including insertions that did not alter insecticidal activity (active) and those that resulted in the loss of insecticidal activity (inactive). Scos, cosmid vector. (B) Map of the insert in the smallest active clone 338/2 constructed using the SalI site at the end of the chitinase gene (chi) and one transposon insertion obtained at the end of the xptA2 gene (dotted lines). (C) Map of insert in plasmid pHRIM801, where the xptA1 gene was expressed from the bacteriophage P_L promoter (P_L).

FIG. 3

Multiple sequence comparison of the protein sequences XptA1 and XptA2 from X. nematophilus and TcbA and TcdA from P. luminescens using the package Plotsimilarity. Scores at amino acid positions along the alignment are presented (Henikoff-Henikoff amino acid similarity score, which ranges from −4 to +11), with the average similarity score represented by a dashed line.

FIG. 4

Map of cHRIM1 indicating the position of mobile elements and areas not related to known insecticidal genes. Restriction sites for SalI and BamHI are shown. transp., hypothetical transposase, retro., retrotransposon; phage, bacteriophage DNA; mit1 and mit2, AT-rich regions similar to mitochondrial DNA; Scos, cosmid vector (size in kilobase pairs).

FIG. 5

SDS-PAGE analysis of the expression of xptA1 from the P_L promoter in E. coli cells. Whole-cell extracts after 18 h of growth at 37°C. Lane 1, E. coli (pHRIM600); lane 2, E. coli. Cell fractions after growth for 4 h at 30°C (lanes 3 and 5) and for 18 h at 30°C (lanes 4 and 6). Samples were separated into inclusion bodies (lanes 3 and 4) and soluble fractions (lanes 5 and 6). Markers (M) are presented (in kilodaltons).