Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Abstract

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.

FIG. 1.

Phylogenetic tree showing distances based on the 16S rRNA sequences obtained from isolates within insect-pathogenic nematodes that infected G. melonella. The analysis was carried out using the programs Dnadist (Jukes-Cantor maximum likelihood) and Neighbor (unweighted-pair group method using average linkages). The scale bar indicates 0.1% estimated sequence divergence. Sequences homologous to that of P. stuartii are indicated by asterisks. The strain shown is one representative of the group whose sequences differ by less than 2 bp.

FIG. 2.

Split decomposition analysis of housekeeping genes with SplitsTree. By this method, evolutionary data are canonically decomposed into a sum of weakly compatible splits and are represented by a splits graph. A, ompR; B, serC; C, asd; D, recA. Each scale bar represents the percent difference among the sequence types. The divergent asd and ompR alleles from strains X. bovienii GS and WSX97a were omitted from the analysis.

FIG. 3.

Bootscanning of nucleotide similarity of different fliC alleles, highlighting their mosaic structure. The graphs show examples of the similarity between the consensus sequence being studied (query sequence) and all other consensus sequences (reference sequences) across the entire length of the fliC gene PCR product. The query consensus sequences for each graph were composed of the following alleles: A, fliC27 and -40; (B) fliC4,-15, -16, and -29; and (C), fliC10 and -39. The reference sequences shown were composed of the following alleles: 1, fliC20, -21, -31, -33, -34, and -41; 2, fliC22 and -30; 3, fliC10 and -39; 4, fliC3, -8, -11, -12, -32, -35, and -36; 5, fliC1, -7, -13, -18, -19, and -23; 6, fliC4, -15, -16, and -29; 7, fliC3, -8, -11, -12, -32, -35, and -36; and 8, fliC5.

FIG. 4.

Structures of the xpt presumptive pathogenicity island in X. nematophila isolates, highlighting differences between the strains. Variations (a to c) in the left- and right-hand junctions of strains 9965, 0014, 0015, N38, L34, QQ2, and PMFI296 are highlighted. The positions of IS630-1 in strain PMFI296 and ISAS-1 in strains 0015, QQ2, N38, and L34 are indicated on the map line. The scale is in bp. ░⃞, AT-rich regions; ▪, core chromosomal genes; ▧, insecticidal toxin genes; ▥, phage-like genes; ⊔, transposon-like genes; □, other genes. Dotted lines show areas of deletion.

FIG. 5.

Comparison of the xpt loci of X. nematophila PMFI296 to those of X. bovienii GS. Areas of colinearity are marked with a dotted line. ▪, core chromosomal genes; ▧, insecticidal toxin genes; ▥, phage-like genes; □, other genes. Note that the homology between the strains extends beyond the core chromosomal genes into the phage-like genes at the RHJ of the putative xpt PAI.

FIG. 6.

Southern hybridization of restriction-digested bacterial chromosomal DNA with digoxigenin-labeled IS probes. (a) HindIII-digested DNA probed with ISAS-1; (b) EcoRI-digested DNA probed with IS630-1; (c) HindIII-digested DNA probed with IS1388; (d) HindIII-digested DNA probed with IS630-2; (e) HindIII-digested DNA probed with TnpA-1; (f) HindIII-digested DNA probed with TnpA-2; (g) HindIII-digested DNA probed with ISPSY-11; (h) HindIII-digested DNA probed with Xis. X. nematophila isolates: 1, L34; 2, N38; 3, QQ2; 4, 0015; 5, 0014; 6, PMFI296; 7, 9965. Markers are λ DNA cut with HindIII.

FIG. 7.

Structure of the xpt genes in X. bovienii isolate I73, highlighting classical features of a type 2 transposon. The same loci on the X. bovienii and P. luminescens chromosomes are also shown for comparison. The sequence of the predicted point of insertion (based on the X. bovienii GS sequence) is shown, with the duplicated target sequence highlighted in boldface. The scale is in bp. ▪, core chromosomal genes; ▧, insecticidal toxin genes; ▥, plasmid-like genes; ⊔, transposon-like genes.