Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens

Abstract

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

FIG. 1

(A) The HindIII fragment from pADAP cloned into pLAFR3 to form pGLA20, showing locations of the mini-Tn10 103 insertion mutations at positions -10, -13, and -35 (18). Results of bioassay of mutants against the grass grub are shown. The map of pBG35 shows the relative position of pGLA20-35 mutation and the location of the 2.2-kb EcoRI fragment used as a probe to screen the pADAP BamHI library. (B) Restriction enzyme maps of the pathogenic clones pMH32 and pMH41. (C) Locations and phenotypes of mini-Tn10 insertions in pBM32. (D) Bioassay results of the pADK recombinants. (E) Schematic diagram of the sequenced region. (F) Nucleotide sequence of the 7-bp repeat, five-copy 12-bp repeat, and the downstream degenerate 34-bp inverted repeat. ∗, pADK mutations isolated by Grkovic et al. (20); filled circles, mutations that resulted in an unaltered pathogenic phenotype (clear gut, nonfeeding); open circles, mutations that resulted in the abolition of pathogenicity; half-filled circles, mutations that induced a nonfeeding pathotype without clearance of the gut; ▿, site of internal deletion; ■, pBR322 vector DNA; □, pLAFR3; ★, location of nucleotide repeats. Arrows indicate ORFs and their orientation. Abbreviations for restriction enzymes: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; X, XbaI.

FIG. 2

Alignment of amino acid sequences of the SepA and P. luminescens toxin components TcbA, TcdA, TcaB, and TccB. ■, RGD motif.

FIG. 2

Alignment of amino acid sequences of the SepA and P. luminescens toxin components TcbA, TcdA, TcaB, and TccB. ■, RGD motif.

FIG. 3

Alignment of amino acid sequences of the SepB, P. luminescens toxin component TcaC, and SpvB.

FIG. 4

(A) Alignment of amino acid sequences of the SepC and P. luminescens toxin component TccC. Conserved positions of the repeat motif GxxRYxYDxxGRL(I/T) (●) are marked. (B) Alignment of amino acid sequences of SepC to the P. luminescens toxin component TccC, the Rhs elements (RshE, P24211; RshD, P16919; RshC, P16918; RshF, I69801; RshB, P16917; RshA, P16916), the hypothetical protein SC2H4.02 from S. coelicolor A3(2), and the wall-associated protein of B. subtilus (WapA). ●, position of the conserved glycine residue which characterizes the junction between the conserved carboxyl end of the Rhs core and the variable carboxyl terminus.

FIG. 5

GC content (window size, 100; window position shift, 3) and hydropathicity plots of SepC, TccC, and RhsD (scanning window of 17 amino acid residues). Each vertical dashed bar denotes the position of the conserved glycine residue which characterizes the junction between the conserved carboxyl end of the Rhs core and the variable carboxyl terminus.