Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica

Abstract

Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) 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 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues.

FIG. 1.

Schematic of pADAP showing the locations of the restriction enzyme sites for HindIII (outer circle), EcoRI (middle circle), and BamHI (inner circle); the previously identified sep virulence gene (sepA, sepB, and sepC)-associated region (23); and the previously constructed pADAP deletion derivatives, pADK93XbaIΔStuRI, pADK93XbaIΔ14XbaI, and pAD32ΔBglII (Table 1). The peripheral semicircles signify areas of pADAP deleted via the use of the designated flanking restriction enzyme sites (22). Corresponding bioassay results are represented by solid (healthy larvae) and shaded (healthy but nonfeeding larvae) semicircles (Table2). The locations of the XbaI restriction enzyme site and the pUH5.4 (crosshatched HindIII fragment) subclone containing the unique AvrII site used to clone the afp cluster are shown (see the text). Restriction enzymes are abbreviated as follows: A, AvrII; B, BamHI; Bg, BglII; H, HindIII; S, StuI; and X, XbaI.

FIG. 2.

Construction of pADAP deletion variant pADS93XbaIΔStuIΔBglIIKn. Shown is a schematic of the previously constructed pADAP deletion variant pADS93XbaIΔStuI with the previously deleted 33.8-kb region (22). The region in the dotted bracket is the 25-kb HindIII fragment in which the 16-kb BglII deletion variant was constructed. To construct a BglII deletion derivative internal to pMH52, the 25-kb HindIII fragment of pMH52 was ligated into the analogous site of pUC19. The resultant construct, pUC52, was restricted with the enzyme BglII and self-ligated (excising 16 kb) to make a vector with a single BglII site, into which the excised BamHI mini-Tn10 kanamycin derivative 103 fragment was inserted, allowing for a selective marker for the recombination of the deletion derivative back into pADAP. The resultant HindIII::mini-Tn10 fragment was then restricted and ligated into the analogous site of pLAFR3. The correct clone was designated pMH52ΔBglII and electroporated into A1MO2(pADS93XbaIΔStuI) for homologous recombination as previously described (see Materials and Methods). Restriction enzymes are abbreviated as follows: B, BamHI; Bg, BglII; H, HindIII; R, EcoRI; S, StuI (only this site is shown); and X, XbaI. Kn indicates the kanamycin antibiotic resistance marker.

FIG. 3.

Construction of pBRminicosAvrII. The 1.6-kb BglII fragment encompassing the cos site of pLAFR3 was excised and ligated into the BamHI site of pBR322 to form pBRminicos. To incorporate the unique AvrII site, the 5.4-kb HindIII fragment from pUH5.4 (Table 1) was ligated into pBRminicos, and the resultant construct was assessed for the correct orientation to allow the later excision of extraneous EcoRI DNA. The correctly oriented construct, called pBRminicosUH5.4, was then digested with EcoRI and self-ligated to produce the construct pBRminicosAvrII containing the unique AvrII site, allowing the insertion of the 37,732-bp AvrII and its XbaI isoschizomer of pADAP to be introduced. Restriction enzymes are abbreviated as follows: A, AvrII; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII. Antibiotic resistance markers: Ap, ampicillin; Cm, chloramphenicol; Tc, tetracycline.

FIG. 4.

Photographs taken on day 9 of a standard bioassay. (A) Larvae fed A1MO2(pADAP⁺) showing distinct amber coloration and absence of feeding, indicated by the unconsumed carrot cube. (B) Healthy feeding larvae. (C) Larvae fed A1MO2(pADK13); the larvae appear healthy but are unable to feed. (D) Larvae fed E. coli(pAF6). (E) Larvae fed 5.6RK(pAF6)(pADAP⁻) showing nonfeeding, glassy-opaque pathotype and absence of gut clearance, indicated by the red arrows. The red box contains a larva in the later stages of the disease before its eventual death.

FIG. 5.

Schematic of the cloned S. entomophila antifeeding gene cluster pAF6. (A) Gene annotation (Table 3), locations of mini-Tn10 insertion points, and results of bioassay (Table 2). The solid circles represent mutations of the pAF6 clone that resulted in an unaltered pathotype (nonfeeding with glassy-opaque appearance); the open circles represent mutations that resulted in the abolition of pathogenicity (P < 0.05). The AvrII and AbaI restriction enzyme sites used to clone the afp cluster are shown. *, peripheral HindIII site from pUH5.4 (see the text). The relative positions of the amb2 locus, phage lysis cassette, and afp cluster are indicated. (B) G+C content of the antifeeding gene cluster (window size, 300; window position shift, 3). The nucleotide numbering is shown below the graph and is relative to the previously annotated sequence listed under accession number AF135182 (dashed line).

FIG. 6.

Predicted genetic organizations of the S. entomophila afp gene cluster and its P. luminescens subsp. laumondii TTO1 analogues (12). The diagram is to scale and is positioned relative to the ORF afp1. ORFs are represented by arrows, with their designations below; the boxes represent remnant elements. Areas of significant protein similarity are depicted by similar shading patterns. The arrows with diagonal lines represent proteins containing a repeat motif; the checkered elements signify similarity to transposon-type elements. Solid stars indicate ORFs in which the translated product has similarity to the cited virulence factor. Open stars indicate tentative virulence factors with no homologues with functional identity in the current databases (see the text). Similarities to protein domains are listed above the afp gene cluster (Table 3). The number at the end of each schematic indicates the nucleotide number of the afp cluster (GenBank accession no. 38176651) or in P. luminescens subsp. laumondii TTO1 (GenBank accession no. 37524032).

FIG. 7.

Alignment of amino acid sequences of Afp2, Afp3, Afp4, and P. luminescens subsp. laumondii TTO1 homologues. Identical amino acid residues are shaded.

FIG. 8.

Alignment of amino acid sequences of Afp13 and P. luminescens subsp. laumondii TTO1 and W14 homologues. Three large repeat sequences (labeled i to iii) were detected within the Afp13 amino acid sequence. Repeats i and iii are 88 amino acid residues long (boxed) compared to the smaller 73-amino-acid degenerate repeat (ii; boldface box). Identical amino acid residues are shaded.

FIG. 9.

Alignment of the carboxyl-terminal amino acid sequences of Afp15 and P. luminescens subsp. laumondii TTO1 homologues showing the locations of the tentative Walker A (GX4GKT) and Walker B (YHyDE) domains, where X is any amino acid and Hy is a hydrophobic amino acid. The solid triangle indicates the location of the pAF6-1 mutation. Identical amino acid residues are shaded.