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 g 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 g 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.Currently, the most successful microbial insecticide is based on the bacterium Bacillus thuringiensis (16, 21) that produces insecticidal crystalline toxins during sporulation (20). The recent use of B. thuringiensis on a large scale and the development and use of transgenic plants expressing these toxin genes (3, 23) may enhance the development of resistant insect populations. New protein toxins are therefore required to provide a greater diversity of genes for use in pest control (14,15,31).In the search for new genes, we have identified, by testing nonluminescent bacterial strains (Xenorhabdus species) isolated from insect parasitic nematodes (IPNs) for their ability to kill insects, a group of orally active protein toxins (22). Both IPNs and even their associated bacteria and toxins when injected into the insect hemocoel are well known for their ability to kill insects (2,4,12,27,32,33). These properties are important for pathogenicity; however, for exploitation a toxin that is active when fed t...

Pathogenicity of additional strains of Photorhabdus and Xenorhabdus (Enterobacteriaceae) to the mushroom mite Luciaphorus perniciosus (Acari: Pygmephoridae)

“…P. luminescens bacteria are known to produce insecticidal proteins (Bowen and Ensign, 1998;ffrench-Constant and Bowen, 2000;Au et al, 2004) and an extracellular protease has been suggested to participate in activation of the toxin complex (Bowen et al, 1999). X. nematophila bacteria also express insecticidal proteins (Morgan et al, 2001) and secrete proteases which are known to be active against insect immune factors (Caldas et al, 2002); however, this and our previous study (Bussaman et al, 2006) are the first to report the miticidal activity of Photorhabdus and Xenorhabdus 297 Symbiotic Bacteria against the Mushroom Mite bacteria. Our results also show that bacterial culture of the X1 strain of X. nematophila is more effective than the previously tested strains Thai and All.…”

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

“…In some cases, expression of individual tcA genes is sufficient to cause toxicity (8,11), but full toxicity generally requires all three Tc components, with the B and C components providing a potentiation of toxicity (12,13). Interestingly, TcB and TcC components coexpressed with TcA components from other species (or from other Tc clusters) can combine to cause an effect with altered host specificity (12,13), suggesting the TcA component is a determinant of target host range.…”

“…Subsequent to their initial discovery, the Tc classes have been redesignated A, B, and C, and the P. luminescens Tc proteins can thus be classified as either TcA-like (TcaA1, TcaB1, TcbA, TccA and TcdA1), TcB-like (TcaC1 and TcdB1), or TcC-like (TccC1 and TccC2) (6). Genes encoding Tc-like proteins have subsequently been identified in many insect-active bacteria including Serratia entomophila (tcA, sepA; tcB sepB; tcC sepC) (7) and the nematode-associated bacterium Xenorhabdus nematophila (tcA, xptA1, xptA2; tcB, xptC1; tcC, xptB1) (8), while the completion of bacterial genome sequencing projects such as Yersinia pestis C092 (9) and Pseudomonas syringae pv. tomato DC3000 (10) has revealed the presence of additional putative insecticidal tc genes.…”

A Novel Secreted Protein Toxin from the Insect Pathogenic Bacterium Xenorhabdus nematophila

“…The high molecular mass complex consists of about 10 polypeptides, ranging in size from 30 to 200 kDa, which is secreted from the bacteria and requires proteolytic processing for activity. Multiple tc genes have been identified in several Photorhabdus species (5,36,37), and tc-like genes have been identified in other insect-associated bacteria (X. nematophila (36,38), Serratia entomophila (39), and Y. pestis (36)), as well as in Pseudomonas syringae pv. tomato, Fibrobacter succinogenes, and Treponema denticola (40).…”