The Main Virulence Determinant of Yersinia entomophaga ...dus nematophilus (60), Pseudomonas...

JOURNAL OF BACTERIOLOGY, Apr. 2011, p. 1966–1980 Vol. 193, No. 80021-9193/11/$12.00 doi:10.1128/JB.01044-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Main Virulence Determinant of Yersinia entomophagaMH96 Is a Broad-Host-Range Toxin Complex

Active against Insects�†Mark R. H. Hurst,1* Sandra A. Jones,1 Tan Binglin,1 Lincoln A. Harper,1

Trevor A. Jackson,1 and Travis R. Glare2

BioControl and BioSecurity, AgResearch, Lincoln Research Centre, Private Bag 4749, Christchurch 8140, Canterbury,New Zealand,1 and Bio-Protection Research Centre, P.O. Box 84, Lincoln University,

Lincoln 7647, Canterbury, New Zealand2

Received 2 September 2010/Accepted 19 January 2011

Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the ento-mopathogenic bacterium Yersinia entomophaga MH96 was localized to an �32-kb pathogenicity island (PAI)designated PAIYe96. Residing within PAIYe96 are seven open reading frames that encode an insecticidal toxincomplex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC componentsbut also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region(�19 kb) of PAIYe96 was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the �TCderivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the �TC mutant straincould be restored by the introduction of a clone containing the entire PAIYe96 TC gene region. As much as 0.5mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detectedin the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but notfrom the �TC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses(LD50s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ngand 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostellalarva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.

Toxin complexes (TCs) active on insects were first identifiedin the nematode-associated bacterium Photorhabdus lumine-scens and termed TCs, as three proteins combined to form acomplex with insecticidal activity (5). TC toxins were subse-quently identified in the genome of Serratia entomophila wherethey reside in the designated gene order sepA, sepB, and sepC(33); this toxin complex ABC designation defines the revisednomenclature of the TC proteins (25). The TC toxins derivedfrom P. luminescens reside as multiple but dissimilar ortho-logues throughout the P. luminescens T011 genome (22), anddifferent insecticidal activities may be attributed to a differentTC cluster (32). The S. entomophila sepABC genes are plasmidborne, and their translated products are host specific, onlycausing amber disease in larvae of the New Zealand grass grubCostelytra zealandica (Coleoptera: Scarabaeidae) (33). TC-liketoxins have since been identified in the genome of Xenorhab-dus nematophilus (60), Pseudomonas syringae pv. tomatoDC3000 (9), and some Yersinia species. The toxin complex A(TCA)-like (tcaB) gene of Yersinia pestis CO92 contains aframeshift mutation, and the toxin complex B (TCB)-like(tcaC) gene contains an internal deletion (51), indicative of aloss of function, while the corresponding TCA-like and TCB-

like orthologues in Y. pestis KIM and 91001 do not (18, 62).Tennant et al. (66) showed that mutations in each of theYersinia enterocolitica biotype 1A T83 genes, TCA-like (tcbA),TCB-like (tcaC), and TCC-like (tccC) genes, resulted in areduced ability to colonize the intestinal tracts of BALB/c micecompared to the wild-type strain. This suggests that the TCproteins may enhance the persistence of the host bacterium, ascenario postulated by Erickson et al. (23), who found noYersinia pseudotuberculosis TC-related toxicity toward the ratflea Xenopsylla cheopis even though expression of the TC genesis upregulated during the disease process. Bowen et al. (5) alsoidentified insecticidal activity when the P. luminescens toxincomplex (TCA) was injected into the hemocoel of Manducasexta.

Although the mode of action of TC proteins has yet to befully elucidated, the expression of individual TC genes hasbeen reported to be sufficient to cause toxicity (45, 50). Fulltoxicity requires all three toxin complex ABC components,with the TCB and TCC components providing a potentiationof toxicity. If the TCB and TCC components are coexpressedin the same cell, they can be combined with TCA componentsfrom other species, or other TC clusters to cause an effect withaltered host specificity, suggesting that the TCA componentrelates to target host range (32, 60, 69). Further evidence forthis was provided by Lee et al. (43), who showed that the X.nematophila XptA1 TCA component is able to bind to brushborder membrane vesicles of Pieris brassicae, and through elec-tron microscopy and three-dimensional (3-D) structural anal-ysis showed that XptA1 forms a tetramer-like structure withwhich the TCB and TCC components are theorized to interact.

* Corresponding author. Mailing address: BioControl and BioSecu-rity, AgResearch, Lincoln Research Centre, Private Bag 4749,Christchurch 8140, Canterbury, New Zealand. Phone: 64 03 3259919.Fax: 64 03 3259946. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 28 January 2011.

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Lang et al. (42) proposed a model in which the P. luminescensTCA component (TcdA1) first makes contact with the targetcell wall, whereby the TCC-type toxins TccC3 and TccC5 areinternalized into the cytosol, inducing actin clustering, throughADP-ribosylation and RhoA GTPase activity, respectively.

Recently, changes in culture temperature have been foundto influence TC gene expression. Through the use of Tn5lux Y.enterocolitica W22703 genome fusions, Bresolin et al. (8) foundthat temperatures below 30°C were optimal for the expressionof the TC genes, TCA-like (tcaA, tcaB1, and tcaB2), TCB-like(tcaC), and TCC-like (tccC) genes, and that sonicated cellextracts, but not culture supernatants, derived from Y. entero-colitica W22703 grown at 30°C showed insecticidal activity to-ward the larva of M. sexta. However, sonicated cellular extractsderived from a strain containing a mutation in tcaB1 alsoexhibited activity toward M. sexta (8), suggesting that the ac-tivity may not be TC related. Hares et al. (31) demonstratedthat the Y. pseudotuberculosis IP32953 strain secretes its TCderivative into the culture medium between 28 and 37°C, witha basal level of expression and secretion at 20°C but no ex-pression at 15°C. However, the TC had no significant activitytoward M. sexta larvae.

The bacterium Yersinia entomophaga MH96 was isolatedfrom a diseased C. zealandica larva, and through host rangetesting, it was shown to have broad-host-range insect activity,affecting a number of coleopteran and lepidopteran specieswith the greatest biological activity against members of theScarabaeidae family (36). After ingestion of this bacterium, thelarvae change from a healthy gray color to a cream color andthen shiny brown in a process that is accompanied by regurgi-tation, acute diarrhea, and then death of the insect within 72 hof infection (M. R. H. Hurst, unpublished data).

In this study, we describe the transposon mutagenesis, nu-cleotide sequence analysis, and identification of a unique clus-ter of TC genes that are the main virulence components of a32-kb pathogenicity island termed PAIYe96 and represent anew member of the TC family. We demonstrate that at tem-peratures at or below 25°C, Y. entomophaga MH96 releaseslarge amounts of the TC into Luria-Bertani (LB) broth, whileat 37°C no TC is released into culture supernatant. By usingultracentrifugation, we could purify the TC. The TC could bevisualized by electron microscopy. The host range activity to-ward a number of insect species of the TC could then beassessed.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. Bacterial strains and plas-mids used in this study are listed in Table 1. Bacteria were grown in Luria-Bertani (LB) broth or on LB agar, and the bacteria were grown at 37°C forEscherichia coli and at 30°C for Y. entomophaga MH96 and its derivatives.Cultures were incubated with shaking at 250 rpm in a Raytek orbital incubator.The antibiotics used for Y. entomophaga MH96 were ampicillin (400 �g ml�1),chloramphenicol (90 �g ml�1), kanamycin (100 �g ml�1), tetracycline (30 �gml�1), and spectinomycin (100 �g ml�1). The antibiotics used for E. coli wereampicillin (100 �g ml�1), chloramphenicol (30 �g ml�1), kanamycin (50 �gml�1), tetracycline (15 �g ml�1), and spectinomycin (100 �g ml�1).

General DNA techniques. Standard DNA techniques, including Southern blot-ting and colony hybridizations were performed as described by Sambrook etal. (59). Plasmids were electroporated into the appropriate Y. entomophagaMH96 strains using a Bio-Rad gene pulser (25 �F, 2.5 kV, and 200 �) (20).pBRminicos2 derivatives were packaged using a Gigapack IIIXL packagingextract (Stratagene). Primers and amplicons used in the study are listed in Table

S1 in the supplemental material. PCR products and plasmid templates werepurified using the high pure PCR product purification kit and the high pureplasmid isolation kit (Roche Diagnostics GmbH), respectively. The pKSAL andpKX3 clones (Table 1) were sequenced at the Australian Genome ResearchFacility (http://www.agrf.org.au/). Sequences were assembled using SEQMAN(DNASTAR Inc., Madison, WI). Databases at the National Center for Biotechnol-ogy Information (NCBI) were searched using BlastN, BlastX, and BlastP (1). Po-tential secretion signal sequences and associated cleavage sites were predicted bySignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/).

Phylogenetic and similarity analyses of YenA2. The protein YenA2 was cho-sen, as evidence suggests that the TCA protein specifies target host range (32, 60,69). Conserved sequences were identified by carrying out a BlastP search againstthe nonredundant protein sequence database at the NCBI. Full-length, or nearlyfull-length, RefSeq sequences returning an E value of 0.0 were selected foranalysis. Protein sequences were aligned with ClustalW (67) and then concate-nated, trimmed, and back translated to nucleotides, and then multiple alignmentwas undertaken using MEGA4 (65). The alignment was cleaned with GBlocks(11), specifying a minimum block length of 5, the presence of an amino acid in50% of sequences to qualify as conserved, and a maximum of 8 contiguousnonconserved sites. Saturation of the resulting sequence of 46 conserved nucle-otide blocks was tested by DAMBE (70). Parameters for the best approximationof nucleotide substitution in this data set were identified by Modeltest 3.8 (55).Phylogenetic relationships were estimated with PHYML (28) using maximumlikelihood with a heuristic search, based on a neighbor-joining starting tree. Therobustness of the topology was estimated with 1,000 bootstrap replicates. Con-sensus trees were drawn with the PHYLIP (24) program consense using theextended majority rule.

Transposon mutagenesis. Transposon mutagenesis was performed using themini-Tn5 derivative Tn5 Km1 as described previously (17). To allow antibioticselection of the recipient strain, the pACYC184 plasmid was transformed into Y.entomophaga MH96. The region including and flanking the transposon insertionpoint was cloned into pUC19 using Tn5 Km1-derived restriction enzyme sites,and the DNA was sequenced using the transposon-specific primers TnR1 andTnPst. Antibiotic markers were recombined into Y. entomophaga MH96 by eitherhomologous recombination, mediated by the suicide vector pVIK165 as de-scribed previously (38), or through pLAFR3-based homologous recombinationas previously described (33). Recombinants were validated by PCR and Southernanalysis.

Cloning the virulence region of Y. entomophaga MH96. Restriction enzymeanalysis identified that the entire PAIYe96 TC-like cluster could be cloned usingthe restriction enzyme PacI (Fig. 1B). The spectinomycin resistance ampliconSPSP (see Table S1 in the supplemental material) was ligated into the SphI siteof pKSALH3 in a region outside PAIYe96 (Fig. 1B). The formed construct(pKSALSp) was digested with HindIII and SalI ligated into the analogous site ofpVIK165 to form pVSALSp (Table 1), allowing subsequent integration of theantibiotic cassette into the Y. entomophaga MH96 chromosome by homologousrecombination to form Y. entomophaga MH96Sp. Y. entomophaga MH96Spgenomic DNA was purified, digested with PacI, ligated to the analogous site ofpBRminicospac, and transduced into the E. coli strain XL1-BlueMRA. Severalspectinomycin-resistant colonies were screened by restriction enzyme analysis,and two constructs designated pPAC12 and pPAC14 of opposing clone orienta-tion were stored.

Construction of a PAIYe96-TC and yenC1 yenC2 and yenUVTW deletion vari-ants. To construct the Y. entomophaga MH96 TC deletion derivative �TC strain,the chloramphenicol resistance amplicon BGCM was cloned into the BglII siteof pPAC14, deleting 19,067 bp of DNA (Table 1 and Fig. 1B) to formpPAC14CM. The pPAC14CM construct was digested with PacI and ligated intothe analogous site of pLAFRP (Table 1), and the chloramphenicol marker wasrecombined into the genome of Y. entomophaga MH96 by homologous recom-bination. To construct a double yenC1 yenC2 mutant, the 1,379-bp EcoRV-flanked spectinomycin resistance amplicon RVSP was ligated into the analogoussite of the pPAC14 EcoRI subclone pPRI (Table 1) to form pPRISP. The EcoRIspectinomycin resistance-containing fragment was then cloned into the analo-gous site of the suicide vector pJP5608 to form pJPRI, which allowed thegeneration of the yenC1 yenC2 �C1-2 mutant by homologous recombination(Fig. 1B and Table 1). Deletion of yenUVTW was accomplished by cloning theNcoI-flanked kanamycin resistance amplicon NCKN into the analogous site ofthe pPAC14 SphI subclone (pPSH). The construct formed (pPSK) was digestedwith SphI, and the kanamycin resistance-bearing fragment was ligated into theSphI site of pJP5608. The correct construct, pJPSK (Table 1), was conjugatedinto Y. entomophaga MH96, allowing the deletion of yenUVTW by homologousrecombination and the formation of the �U-W strain.

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Purification of Y. entomophaga MH96 TC. From a 50-ml overnight culturegrown at 25°C, bacterial debris was removed by centrifugation (10 min, 10,000 � g,4°C) followed by filter sterilization of the supernatant through a 0.2-�m SartoriusMinisart filter into a sterile tube. A 4-ml aliquot of the supernatant was centri-fuged (2 h, 250,000 � g, 4°C) in a Sorvall RZ M120EX ultracentrifuge. The pelletwas resuspended in 100 �l of 0.5� LB broth and further centrifuged (10 min,12,000 � g, 4°C). The supernatant was applied to the surface of a 4.5-ml step

gradient (1.0 ml of 5% glycerol layered on top of 3.5 ml of 40% glycerol in 0.5�LB broth) and centrifuged (2 h, 250,000 � g, 4°C). The pellet was finally resus-pended in 0.25 ml of TM buffer (0.05 M Tris [pH 7.5], 0.1 M MgSO4 � 7H2O).

Protein analysis and chitinase assay. Protein concentrations were determinedbased on the method of Bradford (6), using the Bio-Rad protein assay kit.Assessment of chitinase activity was undertaken using the Sigma Aldrich chiti-nase assay kit, which uses an assay based on the release of p-nitrophenol, from

TABLE 1. Bacterial strains and plasmids used in this study

Bacterial strain orplasmid Relevant characteristics Reference or

source

E. coli strainsDH10B F� mcrA �mrr-hsdRMS-mcrBC�80d lacZ�M15 �lacX74 endA1 recA1 deoR �ara leu-7697 araD139

galU galK nupG rpsL ��47

S17-1 �pir hsdR Pro �recA RP4-2 Tc::Mu Kn::Tn7 integrated in the chromosome of the pir gene 49XL1-BlueMRA �mcrA183 �mcrCB-hsdSMR-mrr-173 endA1 supE44 thi-1 reA1 gyrA96 relA1 Stratagene

Y. entomophaga strainsMH96 Type strain; isolated from a diseased C. zealandica larva 36MH96::1 Tn5 Km1 insertion 749 bp 3� of the initiation codon of yenB; Knr This studyMH96::2 Tn5 Km1 insertion 739 bp 3� of the initiation codon of yenB; Knr

MH96::3 Tn5 Km1 insertion 3,049 bp 3� of the initiation codon of yenA2; Knr






MH96::9 Tn5 Km1 insertion 742 bp 3� of the initiation codon of yenB; Knr



MH96::12 Tn5 Km1 insertion 9,375 bp 3� of the initiation codon of chi1; Knr

MH96::13 Tn5 Km1 insertion 1,557 bp 3� of the initiation codon of chi2; Knr

MH96Sp MH96 with a spectinomycin resistance cassette recombined into the SphI site of yen10; Spr This study�TC strain MH96 with 19,067-bp BglII deletion and with yenA1 yenA2 chi2 yenB yenC1 yenC2 genes; Cmr This study�C1-2 strain MH96 with 2,649-bp EcoRV deletion and yenC1 yenC2 genes; Spr This study�U-W strain MH96 with 5,943-bp NcoI deletion and with yenU yenV yenT yenW genes; Knr This study

PlasmidspAYC177 Amr Knr 12pACYC184 Cmr Tcr 12pBRminicos2 pBR322 containing pLAFR3-derived BglII cos site inserted into its BamHI site; Amr 34pBRminicospac pBRminicos2 with kanamycin resistance gene flanked by PacI- and EcoRI-flanked restriction

enzyme sites inserted into the EcoRI site; Amr KnrThis study

pHP45 Contains spectinomycin-resistant � fragment; Apr Spr 56pJP5608 R6K-based suicide plasmid; Tcr 52pJPSK pJP5608 containing the SphI kanamycin-resistant fragment from pPSH; Tcr Knr This studypJPRI pJP5608 containing the EcoRI spectinomycin-resistant fragment from pPRISP; Tcr Spr This studypK3X Genomic XbaI 18,243-bp clone; derived from strain MH96::6; Knr This studypKSAL Genomic SalI 23,194-bp clone; derived from strain MH96::9; Knr This studypKSALH3 pKSAL digested with HindIII and self-ligated; Amr This studypKSALSp pKSALH3 digested with HindIII and SalI and ligated into the analogous sites of pVIK165; Knr Spr This studypLAFR3 pRK290 with cos, lacZ, and multicloning site from pUC8; Tcr 63pLAFR3P pLAFR3 with kanamycin resistance cassette flanked by EcoRI and PacI restriction enzyme sites

inserted into the EcoRI site of pLAFR3; TcrThis study

Mini-Tn5 Km1 Mini-Tn5 derivative; Amr Knr 17pPAC12 MH96Sp 41,147-bp PacI fragment in the PacI site of pBRminicospac; clone with TC virulence

genes; Amr SprThis study

pPAC14 Opposing PacI clone orientation to pPAC12; clone with TC virulence genes; Amr Spr This studypPAC14�Bg pPAC14 containing a chloramphenicol resistance cassette inserted into the BglII sites deleting a

19,067-bp BglII fragment; Amr Spr CmrThis study

pPRI pPAC14 with 6,565-bp EcoRI-derived fragment in pUCM19; Cmr This studypPRISP pPRI containing an EcoRV-flanked spectinomycin cassette in the EcoRV site, encompassing

yenC1-yenC2; Cmr SprThis study

pPSH pPAC14 13,667-bp SphI-derived fragment in pUCM19; Cmr This studypPSK pPSH containing a kanamycin NcoI-flanked cassette in the NcoI site; Cmr Knr This studypUCM19 Contains lacZ and multicloning site; Cmr 64pUC19 Contains lacZ and multicloning site; Apr 71pVIK165 Suicide plasmid; Knr 38pVSALSp pKSALSp, digested with HindIII and SalI and ligated into the analogous sites of pVIK165; Knr Spr This study

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a chitinase substrate (the substrates for exochitinase activity were 4-nitrophenyl-N,N�-diacetyl--D-chitobioside and 4-nitrophenyl-N-acetyl--D-glucosaminide--N-acetylglucosaminidase; the substrate for endochitinase activity was 4-nitrophe-nyl -D-N,N�,N�-triacetylchitotriose) which upon ionization in basic pH can bemeasured colorimetrically at 405 nm. Both assays were undertaken in accordancewith the manufacturer’s instructions, and measurements were recorded by aFLUOstar Optima (BMG Labtech) plate reader. Standard sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as de-scribed previously (41). Proteins were visualized by silver staining by the methodof Blum et al. (4). Western immunoblotting was performed by the method ofBlake et al. (3), using New Zealand White rabbit polyclonal antibodies raisedagainst peptides (Mimotopes, Pty Ltd.) based on the amino- and carboxyl-terminal amino acid sequences (YenA1, H-MDKYNN-C-GDSDNV-OH; YenB,MQNSQE-C-DTAALAI; YenC1, H-MNQFDS-C-TVVKLR–OH; YenC2, MD

IQLFS-C-LKRRKSF) linked at the central cystine residue to keyhole limpethemocyanin as the carrier.

Protein peptide analysis. Protein bands were excised from Coomassie brilliantblue-stained SDS-polyacrylamide gels, submitted to trypsin digestion, and pre-pared for liquid chromatography-electrospray ionization ion trap-tandem massspectrometry (LC-ESI-MS/MS) as previously described (53). LC-ESI-MS/MSwas performed at the School of Biological Sciences, Victoria University, NewZealand (http://www.victoria.ac.nz/sbs/). Protein identities were assigned usingMascot (version 2.1.03; Matrix Science) using the NCBI nonredundant proteindatabase (see Table S2 in the supplemental material for GenBank accessionnumbers). For N-terminal sequencing, bands containing 2 to 10 pmol of proteinwere analyzed by Edman degradation using an Applied Biosystems 494 Prociseprotein sequencing system and the “Pulsed Liquid PVDF” sequencing method atthe Australian Proteome Analysis Facility (APAF Ltd.; http://www.proteome.org

FIG. 1. (A) G�C content of PAIYe96 and the associated genomic region (window size 300, window position shift). (B) Schematic of Y.entomophaga MH96 PAIYe96. Black arrows denote Y. entomophaga MH96 genomic DNA. The locations and designations of the PAIYe96 virulencedeterminants as defined in Table 2 are shown. The location of the SphI genomically integrated spectinomycin resistance cassette (Sp) to form Y.entomophaga MH96Sp is shown (Table 1; see text); the PacI restriction enzyme sites used to clone the region associated with the virulence genesare indicated. The BglII, EcoRV, and NcoI restriction enzyme sites used to delete the virulence gene-associated region in the �TC mutant strainand associated yenC1-yenC2 (-�C1-2) and yenUVTW (-�U-W) ORFs, respectively, are depicted (see text). The pKSAL and pKX3 sequencingclones (Table 1) are indicated. (C) Similarity of the toxin complex (TC) YenA1 and YenA2-chitinase associated region to its closest orthologuesof P. luminescens TTO1 (Table 2) and comparison of the relative size of the YenA1 and YenA2 proteins to the toxin complex A (TCA) S.entomophila SepA orthologue. Regions of amino acid similarity to SpvA and SpvB are indicated. Predicted protein domains: YenA1 (SpvA, aminoacids [aa] 56 to 185); YenB, (SpvB, aa 9 to 366; RCC1, aa 572 to 582; RGD, aa 836 to 838); Chi1 (Chi 18 hydrolytic site, aa 248 to 256); Chi2 (Chi18 hydrolytic site, aa 431 to 349); YenC1 (necrotizing factor Cnf1, aa 815 to 819, and its associated Rho-activating domain and catalytic triad [10][underlined] is indicated). The vertical lines denote the conserved amino terminus and the stippling in the arrows indicates the variable carboxylregion of the TCC-like Rhs elements (33). (D) Nucleotide sequence of the Y. entomophaga MH96 (yenA1-yenA2) and P. luminescens TTO1(plu2460-plu2459) translational coupling DNA sequence. The termination codon of yenA1 or plu2460 is shown in italic type. The 5� end of theyenA2 or plu2459 ORF is shown in bold type. The predicted yenA2 or plu2459 ribosomal binding site is underlined. (E) tRNAGly 76-bp directrepeats (DRL76 and DRR76). (F) Nucleotide sequence of the eight-copy 8-bp repeat located upstream of yenU.



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.au). Potential protein coiled-coil regions were predicted by Coiled-coilpredictions server (http://npsa-pbil.ibcp.fr/cgi-bin/primanal_lupas.pl).

Assessment of the culture supernatants and sonicated filtrates for insectactivity and assessment of the effect of temperature on TC stability. Cultureswere independently grown at 20, 25, 30, and 37°C to an optical density at 600 nm(OD600) of 0.6, and the cell pellets and culture supernatants were assessed bySDS-PAGE. For sonication, the cells were harvested by centrifugation (5 min,8,000 � g, 4°C) and the pellet was resuspended in 1.2 ml of phosphate-bufferedsaline (10 mM sodium phosphate buffer [pH 7.4], 2.7 mM KCl, 137 mM NaCl).Two 0.7-ml samples were transferred to a 1.7-ml microcentrifuge tube andsubjected to three 20-s rounds of sonication on wet ice using a Sanyo Soniprep150 sonicator (18 �). The sonicated samples were centrifuged (3 min, 16,000 �g, 4°C), and the supernatant was filter sterilized by passage through a 0.2-�mSartorius Minisart filter to a sterile tube. To determine the effect of temperatureon the toxin, 500-�l aliquots of culture filtrate derived from a culture grown at25°C were subjected independently to 25, 35, 42, 45, 50, 55, 60, and 70°C for 15min and then assessed for loss of insecticidal activity by the standard bioassayagainst C. zealandica larvae.

Transmission electron microscopy. To visualize the TC, 3 �l of the TC prep-aration obtained after ultracentrifugation was applied to Formvar-coated 300-mesh copper grids. The sample was left for 5 min, and excess fluid was drawn offwith absorbent filter paper. A drop of 2% phosphotungstic acid was then addedto each grid, and the excess fluid was drawn off with absorbent filter paper. Thegrids were air dried and examined with a Hitachi H-600 electron microscope (80kV) at a magnification of �150,000.

For assessment of outer membrane vesicle (OMV) formation by bacteria,bacteria (1.5 ml) from an overnight culture were pelleted by centrifugation (10min, 4,000 � g, 4°C) and resuspended in 1.5 ml of sterile distilled water (dH2O).The sample was diluted 1:100 in a solution of 2% gluteraldehyde in 0.1 Mphosphate buffer (0.65 mM K2HPO4, 0.35 mM KH2PO4) at pH 7.2, and thesample was left for 2 h. An aliquot of the solution was then added to analuminum holder and dried under vacuum. The samples were sputter coated withgold-palladium and then examined using a JEOL JSM 7000F field emission gunscanning electron microscope (5 kV) at a working distance of 15 mm.

Bioassay and dose response. Bioassays of the TC against the larvae of the NewZealand grass grub (Costelytra zealandica), redheaded cockchafer (Adoryphoruscouloni), and the Tasmanian grass grub (Acrossidius tasmania) were performed.Healthy second- or third-instar larvae collected from the field were individuallyfed squares of carrot that had been rolled on a lawn of bacteria grown overnighton LB agar, resulting in approximately 107 cells per carrot cube. In the case oftoxin, 5 �l (100 ng) of filtrate sample was applied to the surface of a 3-mm3 carrotpiece, from which the larvae would feed. Twelve second- or third-instar larvaewere used for each treatment. Inoculated larvae were maintained at 15°C inice-cube trays or, for A. couloni larvae, in 12-well Corning Costar cell cultureplates. The larvae were fed treated carrot for 5 days, and signs of disease werenoted.

For neonate larvae of the codling moth (Cydia pomonella), light brown applemoth (Epiphyas postvittana), cotton bollworm (Helicoverpa armigera), and thecluster caterpillar (Spodoptera litura), 20 �l of TC (approximately 400 ng) wasapplied to an air-dried section (10- by 10- by 3-mm3) of general purpose labo-ratory diet mixture (61), allowing the solution to absorb and rehydrate thematrix. In the case of the diamondback moth (Plutella xylostella), filter-sterilizedculture supernatant was diluted 50% in 0.04% Duwett (Elliot Chemicals Ltd.),and 15 �l of the dilutant was spread on a 2-cm-diameter cabbage leaf disc. Theleaf was air dried for 2 h at ambient temperature, and the leaf disc was thenplaced on a piece of Whatman filter paper (grade no. 5) located on the bottomof a small plastic bottle. Six second- or third-instar P. xylostella larvae were addedper treatment. Lepidopteran larvae were maintained on the laboratory bench atambient temperature (approximately 20°C).

To determine the 50% lethal doses (LD50s) of the TC against C. zealandicaand P. xylostella larvae, a 10-fold dilution series of filter-sterilized culture super-natant was set up in 0.1 M phosphate buffer. A 5-�l aliquot of each dilution wasinoculated onto the diet the insect was being fed and assessed at day 5 for relativeefficacy. To assess the ability of the TC to affect Galleria mellonella larva byhemocoelic injection, 10-fold serial dilutions of the filter-sterilized culture su-pernatant in 0.1 M phosphate buffer were made, and 10 �l of each dilution wasinjected into the leg of the third segment of the insect larva.

In all cases, the treatments were randomized, the bioassays were performed atotal of three times, and the insects were observed daily for progression ofdisease. Negative controls had a similar treatment applied but using the TCdeletion variant �TC strain. On day 3, any remaining food was removed andreplenished with fresh untreated food. Sample sizes are shown below in tables.

Sectioning and staining. Larvae from bioassays were chilled on ice (20 min)and then fixed in Bouin’s fluid (24 h). Several incisions were made into the cuticleof first-instar larvae allowing penetration of the fixative. Following fixation,larvae were sent to Gribbles Veterinary Pathology New Zealand for sectioning.Mounted sections were examined by light microscopy with an Olympus BX50light microscope.

Nucleotide sequence accession number. The nucleotide sequences were de-posited in GenBank under accession number DQ400808.

RESULTS

Identification of the Y. entomophaga MH96 virulence deter-minants. In total, 1,400 independent Y. entomophaga MH96mini-Tn5 Km1 mutants were screened for loss of virulenceactivity toward C. zealandica larvae by standard bioassay. Thir-teen avirulent mutants were identified, and DNA sequenceanalysis of the cloned mini-Tn5 Km1 genomic DNA junctionpoints identified that 11 of the mutations were located in TCAand TCB-like toxin complex genes, while the other two muta-tions were located in DNA with BlastX similarity to chitinaseenzymes (Table 2). To complete DNA sequence analysis of thevirulence determinants, two clones, pK3X and pKSAL, wereconstructed using Y. entomophaga MH96::6 and Y. ento-mophaga MH96::9 (Table 1) genomic DNA and the restrictionenzymes XbaI and SalI, respectively (Fig. 1B) and their DNAwas sequenced. In total, 47,476 bp was sequenced and depos-ited in GenBank under accession number DQ400808.

Sequence analysis of the virulence region of PAIYe96. Anal-ysis of the completed DNA sequence of the Y. entomophagaMH96 virulence-associated region identified the presence oftwo TCA-like genes (yenA1 and yenA2), a TCB-like gene(yenB), and two TCC-like genes (yenC1 and yenC2) that resideon a 31,898-bp pathogenicity island (PAI) flanked by twotRNAGly 76-bp direct DNA repeats, designated DRL76 andDRR76 (Fig. 1B and E and Table 2). The PAI has a G�Ccontent of 43.8%, which is lower than the predicted genomecontent of G�C, which is 49.3% (36), and has been designatedPAIYe96. Located 118 bp 5� of DRR76 is a region of DNA withsimilarity to tRNA-Lys and tRNA-Arg (Table 2). An eight-copy 8-bp repeat is located 440 bp upstream of yenU (Fig. 1F).Positioned between the yenC2 and yenU open reading frames(ORFs) is a region of DNA with disjointed BlastX similarity toan S-adenosylmethionine (SAM)-dependent methyltrans-ferase (Table 2), which in Y. enterocolitica subsp. enterocolitica8081 is located juxtaposed to tRNA-Pro. However, one region(bp 38890 to 38935) shares 90% DNA identity to the remnanttransposase subunit B of transposon IS911 (Table 2).

The TCA-like genes are divided into two components, yenA1and yenA2 and, similar to the P. luminescens TTO1 gene cluster(plu2461-plu2458), are located juxtaposed to two chitinase-encoding genes. The translated products of yenA1 (130 kDa)and yenA2 (156 kDa) align successionally to span the entireregion of their larger TCA orthologues such as the 262-kDaSepA protein (Fig. 1C). DNA sequence analysis identified thatthe initiation codon of yenA2 is positioned five nucleotidesupstream of the termination codon of yenA1; a similar scenariowas identified in the P. luminescens TTO1 (plu2460-plu2459)DNA sequence (Fig. 1D). Phylogenetic analysis of YenA2showed that it was most similar to the TccB2 (Plu2459) and theX. nematophilus TC protein XptD1 (Fig. 2). The predictedChi1 and Chi2 proteins have high similarity to chitinases from



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TA

BL

E2.

Similarities

ofproducts

ofputative

OR

Fs

totranslated

amino

acidsequences

inthe

NC

BI

databasedetected

usingB

lastP

PutativeO

RF

product(size

no.ofam

inoacids�)

No.of

bpSim

ilarity,locustag,protein

domain

(size aa�) a

Organism

Accession

no.

Yen1

(1,522)84–4,652

85/91//1520(1–1521),Y

intA_01003310,C

OG

2202(1,522)

Yersinia

intermedia

AT

CC

29909Z

P_04635957Y

en2(319)

4,649–5,60587/93//315

(1–315),YintA

_01003311C

OG

3706;responseregulator

containingC

OG

3706(319);C

heY-like

receiverdom

ainand

aG

GD

EF

domain

Y.interm

ediaA

TC

C29909

ZP_04635956

Yen3

(180)5,920–6,459

91/94//179(1–179),hypotheticalprotein

YPK

_2500(179)

Yersinia

pseudotuberculosisY

PIIIY

P_001721230Y

en4(232)

7,381–6,68390/93//232

(1–232),pseudouridylatesynthases,23S

RN

A-specific

CO

G0564

(232)Y

ersiniam

ollaretiiAT

CC

43969Z

P_04639205D

RL

76

7,877–7,95297%

b(489–504)

tRN

AG

lyE

scherichiacoli

X04176

Yen5

(122)7,978–8,343

69/79//119(1–119),hypotheticalprotein

yrohd0001_21470F

els-1prophage

protein-like;pfam05666

Yersinia

rohdeiAT

CC

43380Z

P_04611936Y

en6(278)

8,767–9,60082/91//276

(1–276),signaltransductionhistidine

kinase(451)

Y.m

ollaretiiAT

CC

43969Z

P_04640870Y

en7(121)

9,962–10,32455/71//119

(2–120),hypotheticalproteinU

TI89_C

4899(122)

E.coliU

TI89

YP_543833

Yen8

(138)10,935–10,519

No

similarity,transcriptionalregulatory

protein,Cterm

inal,pfam00486

Chi1

(543)11,709–13,337

55/71//551(2–541),unnam

edprotein

productPlu2461

(544);putativeexochitinase,C

hiA-like

CO

G3325,pfam

00704.13P

hotorhabduslum

inescenssubsp.

laumondiiT

TO

1C

AE

14835

40/57//517(29–519),exochitinase

CB

M_5_12

(695)Sodalis

glossinidiusC

AA

7220127/43//454

(62–503),OR

F42;C

hiAchitinase,(564)

Spodopteralitura

nucleopolyhedrovirusA

BS70983

35/49//329(251–560),C

hiA,endochitinase

(563)Serratia

marcescens

NP_258310

39/53//524(30–518),C

hi2(634)

Y.entom

ophagaM

H96

AB

G33867

YenA

1(1,165)

13,423–16,91755/71//1183

(1–1164),insecticidaltoxincom

plexprotein

TccA

2Plu2460

(1,173)P

.luminescens

subsp.laumondiiT

TO

1C

AE

1483429/50//134

(187–316),virulenceprotein

SpvA(187)

Salmonella

entericasubsp.arizonae

AA

D02678

YenA

2(1,361)

16,910–21,00468/81//1376


plexprotein

TccB

2Plu2459

(1,362)P

.luminescens

subsp.laumondiiT

TO

1C

AE

14833C

hi2(634)

21,102–23,00372/82//627

(1–627),unnamed

proteinproduct

Plu2458(622),putative

chitinaseC

OG

3325;pfam00704

P.lum

inescenssubsp.laum

ondiiTT

O1

CA

E14832

38/52//523(110–616),C

hi1(543)

Xenorhabdus

nematophila

AB

G33870

54/67//655(4–648),C

hi(648)Y

.entomophaga

MH

96C

AC

38398

YenB

(1,488)23,198–27,661

59/74//1488(1–1483),insecticidaltoxin

complex

proteinT

caC(1,485)

P.lum

inescensA

AL

1845146/60//358

(9–366),SpvB(591)

Salmonella

entericasubsp.enterica

serovarC

holeraesuisN

P_073228

YenC

1(975)

27,757–30,68159/73//679

(8–683),TccC

1/XptB

1protein

(1,016)X

enorhabdusnem

atophilaA

AS45281

31/46//260(699–948),Pnf,R

ho-activatingdom

ainof

cytotoxicnecrotizing

factor(287)

P.lum

inescensA

AN

64212

YenC

2(971)

30,903–33,81570–79//690

(1–690),PTB

2233,putativeinsecticidaltoxin

complex

(994)Y

.pseudotuberculosisIP

32953C

AH

2147148/63//1003


plexprotein

TccC

6(965)

P.lum

inescenssubsp.laum

ondiiTT

O1

NP_931365

33,930–34,25054–70//48, b

remnant

DN

Asequence

ofSA

M-dependent

methyltransferases

Y.m

ollaretiiAT

CC

43969E

EQ

11137

YenU

(857)34,810–37,380

27/467//287(546–795)

hypotheticalproteinPfl_2644

(1,288)P

seudomonas

fluorescensPfO

-1A

BA

7438546/65//145

(140–283)hypotheticalprotein

ET

A_11720

Erw

iniatasm

aniensisE

t1/99Y

P_001907112

YenV

(151)38,263–37,811

79/88//134(16–149)

hypotheticalproteinE

CA

2267(153),pfam

08332.1K

lebsiellapneum

oniaeC

AQ

76722Y

enT(64)

38,757–38,94845/58//51

(1–51)Y

ersiniaheat-stable

enterotoxintype

B(71)

Y.enterocolitica

P7497738,890–38,934

90%b

(1208–1248)insertion

sequenceIS911v4

E.coli

AJ507493

YenW

(95)39,412–39,125

No

similarity

39,640–39,71880%

b(3102–3183)

bacteriophageP27

tRN

A-Ile-tR

NA

-Arg

E.coli

AJ249351

DR

R76

39,850–39,92597%

b(489–504)

tRN

AG

lyE

.coliX

04176Y

en9(599)

41,182–42,97867/75//595

(4–598)E

SA_01767

(604)E

nterobactersakazakiiA

TC

CB

AA

-894A

BU

77021Y

en10(1,211)

42,988–46,62076/84//1207

(1–1206)E

SA_01768

(1,213)E

.sakazakiiAT

CC

BA

A-894

AB

U77022

Yen11

(233)46,655–47,353

70/83//221(8–228)

ESA

_0176901769

(243)E

.sakazakiiAT

CC

BA

A-894

AB

U77023

aPercentidentities

andpercentsim

ilaritiesw

erecalculated

inrelation

tothe

deducedgene

productsofthe

sequencedO

RF

.The

positionsofam

inoacid

similarity

(percentidentity/percentsimilarity

overthe

indicatedrange

ofam

inoacid

residuepositions

shown

inparentheses)

inrelation

tosequence

generatedin

thisstudy

areshow

n;forexam

ple,85/91//1520(1–1521)

means

thatthere

was

85%identity

and91%

similarity

overthe

indicatedresidues.aa,am

inoacids.

bPercent

DN

Asim

ilaritycom

paredto

orthologue.SeeF

ig.1Bfor

schematic

ofY

.entomophaga

MH

96virulence-associated

region.



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Serratia marcescens and the insect symbiont Sodalis glossinidiusand contain a Chi18 hydrolytic motif (68) (Table 2).

The yenT ORF located within PAIYe96 encodes a 64-amino-acid protein with high similarity to heat-stable enterotoxins ofE. coli and Yersinia spp. (Table 2), some of which activateguanylate cyclase within the host intestinal epithelial cells, re-sulting in water loss (21). Amino acid alignment of these pro-teins identified a high degree of amino-terminal similarity pre-ceding a predicted signal cleavage site at residue 23 of YenT.However, no similarity to the enterotoxin carboxyl-based“toxin domain” was identified (see Fig. S1 in the supplementalmaterial). A putative two-component regulator Yen6 is posi-tioned 5� to the PAIYe96 TC gene cluster, while another puta-tive regulator, Yen2, is located external to PAIYe96 5� toDRL76 (Fig. 1B and Table 2). Six additional ORFs comprisethe remainder of PAIYe96, four of which have no significantidentity to protein sequences in the current database (Table 2).

Defining the compound active on insects. Bioassays of theTC gene-bearing pPAC12 and pPAC14 clones in an E. colibackground and the Y. entomophaga MH96 �TC strain (Table1) toward C. zealandica larvae were performed, and the toxinswere found to be innocuous, causing no symptoms of disease(Table 3). However, the ability to cause virulence could berestored to the �TC strain by transforming the bacterium witheither of the TC gene-bearing pPAC12 or pPAC14 clones(Table 3). It is of interest that no Tn5 Km1-based insertionswere identified in the yenC1, yenC2, and yenUVTW ORFs. Toascertain the roles of these genes in virulence, yenC1 yenC2and yenUVTW deletion variants were constructed (as outlined

in Materials and Methods). Bioassays of both Y. entomophagaMH96 �C1-2 and �U-W strains against C. zealandica larvaeshowed abolition and no alteration in virulence, respectively(Table 3).

FIG. 2. Phylogenetic analysis consensus tree for YenA2 sequence to its closest orthologues. The tree is based on 3,515 trimmed nucleotides.The robustness of the topology was estimated with 1,000 bootstrap replicates. Bootstrap proportions are indicated at the branches. GenBankaccession numbers are given in parentheses. The tree was drawn with Treedyn (15).

TABLE 3. Oral toxicity of Y. entomophaga and E. coli strains toC. zealandica larvae on day 5

StrainaMortality (n)b

Significance level ofthe differencec

Treated Control

MH96 36 (36) 0 (36) �0.000 (2.2 � 10�16)MH96Sp 36 (36) 0 (36) �0.000 (2.2 � 10�16)MH96::4 0 (36) 0 (36) NS (1.000)MH96::6 3 (36) 0 (36) NS (0.2394)MH96::9 0 (36) 0 (36) NS (1.000)MH96::12 2 (36) 0 (36) NS (0.4930)MH96::13 0 (36) 0 (36) NS (1.000)�TC strain 0 (36) 2 (36) NS (0.4930)�TC(pPAC12) 36 (36) 0 (36) �0.000 (2.2 � 10�16)E. coli(pPAC12) 0 (36) 0 (36) NS (1.000)�TC(pPAC14) 36 (36) 0 (36) �0.000 (2.2 � 10�16)E. coli(pPAC14) 0 (36) 0 (36) NS (1.000)�C1-2 strain 1 (36) 0 (36) NS (1.000)�U-W strain 36 (36) 0 (36) �0.000 (2.2 � 10�16)

a Y. entomophaga strains MH96 and derivatives (e.g., MH96::4), �TC strain,�C1-2 strain, and �U-W strain and E. coli carrying pPAC12 were studied.

b Mortality is given as the number of larvae killed. n is the sample size in thetreated group or in the negative-control group.

c �0.000 significant difference at a 0.1% level (� 99.9% confidence level). NS,not significant.



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The TC is released into LB broth at temperatures equal toor less than 25°C. Assessment of the culture supernatantsderived from Y. entomophaga MH96 grown in LB broth attemperatures of either 20, 25, 30, and 37°C, in conjunction withSDS-PAGE and bioassay analyses, identified that culture su-pernatants derived from cultures grown at 25°C or less con-tained large amounts of TC active on insects, with prominentYenA1 and YenA2 bands visualized by SDS-PAGE (Fig. 3).Analysis of the bacterial cell pellet by silver-stained SDS-PAGE detected significant levels of the TC componentsYenA1 and YenA2 at 25°C, but visually, only small amountswere seen for cultures grown at 30 and 37°C (Fig. 3). Assess-ment of the sonicated filtrates derived from cultures grown ateither of these temperatures showed that they all had activitytoward C. zealandica larvae, while sonicated filtrates derivedfrom the �TC strain prepared under the same conditions wereinnocuous (Table 4).

Results of host range testing of the culture filtrate of Y.entomophaga MH96 grown at 25°C identified that 5 �l (100 ng)of culture filtrate had high insecticidal activity toward C. zeal-andica, A. couloni, A. tasmania, and P. xylostella larvae and thatculture supernatants derived from the �TC strain preparedunder similar conditions were innocuous (Table 5). Subjectingthe culture supernatant to different temperatures showed thatthe culture filtrate lost insecticidal activity at a temperatureequal to or greater than 60°C (Table 6). C. zealandica larvaefed with the purified TC became diarrhetic and were regur-gitated from the C. zealandica larval mouth parts. The larvaeexhibited reduced feeding activity and changed from ahealthy gray color to a white-amber color within 12 to 18 hafter ingestion; during this time, some of the larvae showed

no clearing of the gut (see Fig. S2 in the supplementalmaterial).

In assessment of P. xylostella larvae, these larvae also exhib-ited reduced feeding activity and the larvae changed to alighter color and became turgid. The third-instar larvae of theredheaded cockchafer (A. couloni) underwent a characteristicamber color change when fed the TC. However, many of thelarvae recovered and later appeared healthy. A repeated doseof the TC at day 3 was needed to induce mortality. Bioassaysof the efficacy of the TC toward the Lepidoptera E. postvittana,C. pomonella, H. armigera, and S. litura showed that there wasno mortality within 5 days of administering the TC to the

FIG. 3. Silver-stained SDS-polyacrylamide gel of Y. entomophagaMH96 culture supernatant (CS) and cell pellets (CP) grown at 25, 30,and 37°C. The migration positions of the YenA1 and YenA2 proteinsare indicated. Lane M contains Bio-Rad broad-range markers. Thepositions of molecular mass markers (in kilodaltons) are shown to theleft of the gel.

TABLE 4. Activity of Y. entomophaga MH96 culture or sonicatedfiltrates to C. zealandica larvae at various

culture temperatures on day 5

Culture or filtrateand strain

Culturetemp(°C)

Mortality (n)aSignificance level of

the differencebTreated Control

Sonicated filtrateMH96 25 34 (36) 0 (36) �0.000 (0.4930)

30 36 (36) 0 (36) �0.000 (2.2 � 10�16)37 31 (36) 0 (36) �0.000 (3.4 � 10�15)

�TC strain 25 0 (36) 0 (36) NS (1.000)30 1 (36) 0 (36) NS (1.000)37 0 (36) 0 (36) NS (1.000)

Culture supernatantMH96 25 36 (36) 0 (36) �0.000 (2.2 � 10�16)

30 1 (36) 0 (36) NS (1.000)37 0 (36) 0 (36) 0 (36)

�TC strain 25 0 (36) 0 (36) NS (1.000)30 0 (36) 0 (36) NS (1.000)37 0 (36) 0 (36) NS (1.000)

a Mortality is given as the number of larvae killed. n is the sample size in thetreated group or in the negative-control group. The negative Y. entomophaga�TC culture supernatant was prepared as Y. entomophaga MH96 was.

b �0.000 significant difference at a 0.1% level (� 99.9% confidence level). NS,not significant.

TABLE 5. Activity of Y. entomophaga MH96 culture filtrates onvarious insect species on day 5

Insect order and speciesMortality (n)a

Significance level ofthe differenceb

Treated Control

ColeopteraCostelytra zealandica 34 (36) 0 (36) �0.000 (2.20 � 10�16)Acrossidius tasmaniae 36 (36) 0 (36) �0.000 (2.2 � 10�16)Adoryphorus coulonic 30 (36) 0 (36) �0.000 (2.37 � 10�14)

LepidopteraEpiphyas postvittana 3 (36)d 1 (36) NS (0.6142)Helicoverpa armigera 2 (36)d 3 (36) NS (1.000)Plutella xylostella 36 (36) 1 (36) �0.000 (2.2 � 10�16)Spodoptera litura 2 (36)d 1 (36) NS (1.000)Cydia pomonella 31 (36)d 2 (36) �0.000 (3.39 � 10�15)

a Mortality is given as the number of larvae killed. n is the sample size in thetreated group or in the negative-control group.


c A. couloni larvae were subjected to a second toxin dose on day 3.d The larvae exhibited reduced feeding and growth.



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larvae, although there was a significant reduction in growthand an absence of frass production (Table 5).

Assessment of the LD50 of the culture supernatant againstlarvae of P. xylostella and C. zealandica showed that as little as30 or 50 ng, respectively, of toxin per larva was sufficient tocause mortality 5 days after ingestion. Some of the C. zea-landica larvae that received a sublethal dose of the TC revertedfrom an amber phenotype to a healthy appearance (Table 7).

Purification and validation of the Y. entomophaga MH96 TCcomponents. Based on the premise that the TC had a com-bined mass of 2.6 million daltons, the Y. entomophaga MH96culture supernatant was pelleted by ultracentrifugationthrough a glycerol step gradient. Assessment of the resus-pended pellet by silver-stained SDS-PAGE enabled 10 prom-inent bands to be visualized (Fig. 4A, UC1P-Y). Six of thesebands, as well as a less-intense high-molecular-weight band(YenB), correlated with the molecular masses of componentsof the TC (Fig. 4A, UC1P-Y). A similar analysis of the ultra-centrifugated pellet of the �TC strain showed no other TC-related bands; however, two bands migrated with the predictedmasses for Chi1 and Chi2. These bands were removed after asubsequent ultracentrifugation through a glycerol step gradient(Fig. 4A, UC2P-�). Four prominent bands of 75, 60, 35, and 32kDa were present in both the �TC strain and Y. entomophagastrain MH96 (Fig. 4A, UC1P), the latter three proteins weresubsequently identified by LC-ESI-MS/MS and/or N-terminalsequence analysis as the GroEL heat shock protein and twoouter membrane-associated proteins, OmpA and OmpC (Ta-ble 8; see Table S2 in the supplemental material). Centrifuga-tion of the resuspended ultracentrifuged pellet through a glyc-erol step gradient (Fig. 4A, UC2P-Y), removed three of theprominent bands, including the GroEL band, although a bandwith a mass similar to that of the GroEL protein remainedin the ultracentrifuged pellet of the �TC strain (Fig. 4A,UC2P-�). With Y. entomophaga MH96, the TC, Chi1, and Chi2proteins sedimented through the gradient (Fig. 4A, UC2P-Y).

LC-ESI-MS/MS analyses identified five of the seven pre-dicted TC-associated proteins: Chi1, YenA1, YenA2, Chi2,and YenB, but not YenC1 or YenC2. Analysis of the �108-kDa band, which was expected to contain comigrating YenC1(109-kDa) and YenC2 (107-kDa) proteins, was unsuccessful inidentifying any peptides from either protein. The most-prom-inent peptides identified matched the amino acid sequences of

the YenA1 and YenA2 proteins. LC-ESI-MS/MS analyses alsoidentified peptides matching the YenA1 and YenA2 sequencesin the 50- to 107-kDa range (Fig. 4C; see Table S2 in thesupplemental material). The YenA1 antibody also cross-re-acted with some of these bands (Fig. 5). Western immunoblot-ting was also able to detect YenB, YenC1, and the YenC2protein (Fig. 5). The amino-terminal peptide of YenC1 wasidentified through N-terminal sequence analysis of the �108-kDa band (Table 8).

Assessment of the N-terminal sequence of the 50-kDa band(Fig. 4C, band 10a,b,c), isolated from three independent Y.entomophaga MH96 cultures grown at 25°C identified threeYenA2 peptide sequences initiating between amino acid resi-dues 767 and 778 of YenA2 (Table 8). Amino acid alignmentof YenA2 to the P. luminescens TCA proteins, TcaB, TcbA,and TcdA, identified a divergent region susceptible to cleavagein all three proteins and coincident with the putative cleavagesite in YenA2 based on N-terminal sequencing (Table 8; seeFig. S3 in the supplemental material). The cleavage-suscepti-ble region is flanked by conserved regions of protein similarityand resides approximately 200 amino acid residues prior to apredicted coiled-coil region located toward the C terminus(YenA2 amino acids [aa] 1025 to 1087). The predicted aminoterminus of YenA2 was identified by N-terminal sequenceanalysis of the �90-kDa band (Table 8).

Analysis of the semipurified pellets derived from ultracen-trifugation of the previously constructed Y. entomophagaMH96 TC mutants by SDS-PAGE showed that Y. ento-mophaga MH96::1, MH96::2, and MH96::9, containing a mu-tation in the 5� region of yenB (Table 1), produced a TC variantcontaining only the YenA1, YenA2, Chi1, and Chi2 compo-nents (Fig. 4B, U2CP-9). No YenB or YenC1 and YenC2protein could be visualized by SDS-PAGE or detected byWestern immunoblotting (Fig. 5). Bioassay of the purifiedYenA1, YenA2, Chi1, and Chi2 complex from Y. entomophagaMH96::9 showed no activity toward C. zealandica larvae (Table3). Assessment of the Y. entomophaga MH96 mutants, Y. en-tomophaga MH96::3-8 and Y. entomophaga MH96::10-13, con-taining mutations in yenA1, yenA2, chi1, and chi2 genes (Table1) showed no complex or chitinases to pellet by ultracentrifu-gation (Fig. 4B, U2CP-7).

Transmission electron microscopy of the ultracentrifuged Y.entomophaga MH96 pellet revealed the presence of outermembrane vesicles (OMVs) ranging in size from 40 to 300 nm,as well as pyramidal structures of approximately 19 nm by 30nm (Fig. 6A); the pyramidal structures were not present in the

TABLE 7. LD50s of semipurified TC on insects on day 5

Insect TC dose(ng)

Mortality (n)aSignificance level of

the differencebTreated Control

C. zealandica 50 17 (36)c 0 (36) �0.000 (1.268 � 10�6)P. xylostella 30 18 (36) 2 (36) �0.000 (3.867 � 10�5)G. mellonellad 35 18 (36) 2 (36) �0.0002


b �0.000 significant difference at a 0.1% level (� 99.9% confidence level).c Some of the larvae reverted to healthy with resumption of feeding.d G. mellonella larvae were injected with semipurified TC into the hemocoel.

TABLE 6. Effect of temperature on Y. entomophaga MH96 culturefiltrates to C. zealandica larvae on day 5

Temp (°C)Mortality (n)a

Significance level of thedifferenceb

Treated Control

25 36 (36) 0 (36) �0.000 (2.2 � 10�16)35 36 (36) 0 (36) �0.000 (2.2 � 10�16)42 36 (36) 1 (36) �0.000 (2.2 � 10�16)45 31 (36) 0 (36) �0.000 (3.4 � 10�15)50 36 (36) 0 (36) �0.000 (2.2 � 10�16)55 28 (36) 0 (36) �0.000 (8.0 � 10�15)60 9 (36) 1 (36) NS (0.014)70 3 (36) 0 (36) NS (0.239)





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�TC strain. Scanning electron microscopy of Y. entomophagaMH96 identified the presence of small and large blebs pro-truding from the bacterial cell wall that typify OMV formation(Fig. 6B).

The semipurified Y. entomophaga MH96 TC was assessedfor both exo- and endochitinase activity and found to have highendochitinase activity but no significant exochitinase (Table 9).Injection of the semipurified TC into the G. mellonella larval

FIG. 4. (A) Silver-stained SDS-polyacrylamide gel of Y. entomophaga MH96 and �TC mutant strain. Y. entomophaga MH96 (Y) and �TCmutant strain (�) in culture supernatant (CS), ultracentrifuged supernatant (UC1S), ultracentrifuged pellet (UC1P), supernatant after ultracen-trifugation and application to a step gradient (UC2S), and pellet after ultracentrifugation and application to a step gradient (UC2P). (B) Silver-stained SDS-polyacrylamide gel of Y. entomophaga MH96::7 (7) and Y. entomophaga MH96::9 (9), showing the absence of formation of thecomplex and a complex missing the YenB component, respectively. (C) SDS-polyacrylamide gel stained with Coomassie brilliant blue showingbands assessed by either LC-ESI-MS/MS (E), N-terminal sequence analysis (N) (Table 8), or Western immunoblot (W). The M lanes containBio-Rad broad-range markers. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. The locations ofcomponents of the TC, GroEL, OmpA, and OmpC proteins are indicated (Table 8; see text; also see Table S2 in the supplemental material).

TABLE 8. Edman N-terminal sequencing data of proteins derived from the ultracentrifuged pellet

Peptide no.a

(band mass kDa�)

NH2-terminal sequenceb

Accession no.Obtained sequence Predicted sequence, protein (kDa)

1 (167) MQN{S}QEM{A}I- MQNSQEMAI, YenB (167) ABG338662 (156) {S}NSIEQKLQ SNSIEAKLQ, YenA2 (156) ABG338684 (107) -N{Q}FL(F/S)ALT- MNQFDSALH, YenC1 (107) ABG338655 (88) SNSIEAKLQ SNSIEAKLQ, YenA2 (156) ABG338689 (57) AAKDVKFGN GroEL (57) YE0354, Yersinia enterocolitica subsp. enterocolitica 8081 YP00100473010a (52) -{Q}PLTF(E/L)PVV QPLTFEPVV, YenA2 (156) ABG3386810b (51) TFEPVVHDQ TFEPVVHDQ, YenA2 (156) ABG3386810c (50) DQTMSSAVDN DQTMSAVDN, YenA2 (156) ABG3386811 (35) APKDNTWYTc OmpA (38) signal sequence cleaved is 36 kDa; YE1581;

Y. enterocolitica subsp. enterocolitica 8081 (357)Y_P001005874

12 (32) AEIYNKDGNc OmpC (39.6) signal sequence cleaved is 37.6 kDa; KPK_1516;Klebsiella pneumoniae (342)

Y_P002237369

a See Fig. 4C for band number.b Braces around a letter indicate that it was a tentative call. -, no call determined due to cycle interference.c N-terminal sequence at the predicted signal sequence cleavage site of its protein orthologue.



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hemocoel showed that 35 ng of the TC was sufficient to give anLD50 within 5 days, while a similar preparation from the �TCstrain was ineffective (Table 7). The larvae showed no initialchange in color but instead became flaccid and eventuallyturned black 6 days after injection. To assess the effects of theTC on the insect gut ultrastructure, 100 ng of TC was fed to P.xylostella larvae, and histology was assessed over a 48-h period.Observations showed that within 24 h of feeding, the intestinalcolumnar cells swelled and cellular vesicles and debris ap-peared in the gut lumen (Fig. 7). Within 48 h, the columnarcells became dislodged, and the midgut epithelial cells becameamorphous with no distinct cell forms being present (Fig. 7D).

DISCUSSION

Through transposon mutagenesis we have defined a31,898-bp pathogenicity island, termed PAIYe96, that containsa cluster of five TC toxin complex genes and two chitinasegenes, the translated products of which are predicted to com-bine to form a large toxin complex (TC). Culture filtratesderived from only Y. entomophaga strain MH96 and not its�TC derivative were able to cause a pathotype similar to thatof the wild-type bacterium.

In cultures where Y. entomophaga MH96 was grown at 25°Cor less, large amounts of the TC were observed in LB broth.However, above 25°C, no toxin was detected in the culturesupernatant. The effect of temperature on bacterial behaviorhas been documented in Yersinia pestis (30) and Yersinia en-terocolitica (8, 14, 46), suggesting a shared phenomenon amongthe Yersinia. As little as 5 �l of filter-sterilized culture super-natant was able to cause lethality in C. zealandica, A. couloni,A. tasmaniae, and P. xylostella larvae (Table 5). The LD50s of

the TC to P. xylostella and C. zealandica larvae were 30 ng and50 ng, respectively. This is significantly lower than the LD50 of875 ng of the P. luminescens-derived toxin A against M. sextaneonates (5). However, this amount correlates with the relativeefficacy of E. coli-expressed Xenorhabdus nematophila XptA1,XptB, and XptC lysates against Pieris rapae (45 ng) or Pierisbrassicae (3.2 ng) or the XptA2, XptB, and XptC TC againstHeliothis virescens (5.3 ng); neither of these TCs was activeagainst P. xylostella (60). Bioassay analysis of the Y. ento-mophaga MH96::9 lacking YenB, YenC1, and YenC2 identi-fied no insecticidal activity when they were fed to C. zealandicalarvae (Table 3), showing that unlike the P. luminescens TcdA(45) and X. nematophilus XptA1 (50) TCA components, theTC-associated chitinases (Chi1 and Chi2), or the TCA (YenA1and YenA2) components are not active on insects.

Not all of the insect species tested were killed by the TC; E.postvittana, C. pomonella, H. armigera, and S. litura exhibitedonly reduced growth when subjected to the toxin. Reducedfeeding activity was also identified by Bowen et al. (5), whoused sublethal doses of the P. luminescens toxin complex(TCA) against M. sexta larvae. A reduction in growth was alsoused as a measure of toxicity of the E. coli-expressed Y. pseu-dotuberculosis IP32953-derived TC toxins, the TCA-like (tcaAtcaB), TCB-like (tcaC), and TCC-like (tccC) toxins, to larvae ofM. sexta (54). Increasing the administered toxin dose or redos-ing the larvae of E. postvittana, C. pomonella, H. armigera, andS. litura was unable to cause mortality (Hurst, unpublished),even though the insects are susceptible to an oral dose of Y.entomophaga MH96 (36). The loss of toxicity could be due toa rapid degradation of the TC in the guts of the insects. Thelarge third-instar larvae of the redheaded cockchafer requireda repeated dose of the TC at day 3 to induce mortality. Asublethal dose of the TC to C. zealandica larvae resulted in areversion effect, similar to that described for the S. ento-mophila-derived Sep proteins (35), which means that a contin-uous supply of the Y. entomophaga TC and TC toxins in gen-eral may be needed to exert an effect. Of further interest, theselarvae underwent a transitional phase where a dark gut couldbe visualized through the cream color, indicating that the guthad not been voided and suggesting that factors other than gutclearance relate to the change in larval coloration.

Transposon mutagenesis identified 13 Y. entomophagaMH96 avirulent mutations that resided in the yenA1, yenA2,

FIG. 5. Western immunoblot of purified Y. entomophaga MH96TC (Y) and the Y. entomophaga MH96::9 TC derivative (9). YenA1,YenB, YenC1, and YenC2 antibodies used as probes are listed at thetop of the lanes in the gel (see Materials and Methods for antibodyprobes). The leftmost three lanes show a Coomassie brilliant blue-stained polyvinylidene difluoride (PVDF) membrane. The small blackarrows point to the migration positions of the YenC1 and YenC2proteins. Lane M, Bio-Rad broad-range marker.

FIG. 6. (A) Transmission electron micrograph of TC and associ-ated OMVs. The white arrows point to TC particles, which appear aselongated pyramid-type structures. The electron micrograph was takenat a magnification of �150,000 (bar � 50 nm). (B) Scanning electronmicrograph of Y. entomophaga MH96 showing OMV blebs taken at amagnification of �15,000 (bar � 1 �m).



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and yenB genes and in the chitinase-encoding genes (chi1 andchi2), confirming the roles of these proteins in insecticidalactivity. However, no mutation was identified in either of theTCC-like (yenC1 or yenC2) components or in other regions ofPAIYe96. Bioassay data of the yenC1 yenC2 double deletionvariant (�C1-2) showed an abolition of virulence, indicatingthat YenC1 and YenC2 may be able to complement the loss ofeach other, a scenario that would explain the inability to detecttransposon insertions in either of these genes by bioassay anal-ysis. The mini-Tn5 Km1 insertions were nonpolar, as the trans-lated products of TC genes located 3� to the insertion pointcould be visualized by SDS-PAGE (see Fig. S4 in the supple-mental material).

Although the pPAC12 and pPAC14 clones with TC geneswere able to complement the loss of the ability to cause viru-lence in the �TC strain, the clones were unable to cause vir-

ulence in an E. coli background. Attempts to artificially inducethe PAIYe96-associated TC cluster have also been unsuccessful(Hurst, unpublished). DNA sequence analysis identified a re-gion of potential translational coupling located between yenA1and yenA2 (Fig. 1D). A similar translational coupling has beendocumented between the lacL and lacM -galactosidase genesfrom the bacterium Lactobacillus lactis (16), where it is sug-gested to enable the stalling and backward movement of theribosomes, allowing the correct translation of the second ORF,i.e., yenA2. This could explain the inability of the clones bear-ing TC genes to cause virulence when in an E. coli backgroundand may indicate that a factor encoded by Y. entomophagaMH96 is needed for correct TC expression.

Ultracentrifugation of the �TC strain or the Y. entomophagaMH96 strain containing mutations in either yenA1 or yenA2showed no sedimentation of the Chi1 and Chi2 proteins. No

FIG. 7. Histopathological effects of TC on the anterior midgut of P. xylostella epithelium. (A) Untreated larva, showing a regulararrangement of basal membrane (bm) and columnar cells (cc) in the midgut epithelium and the lumen (lu). (B to D) Larvae at 16 h (B),24 h (C), and 40 h (D) after the larval ingestion of food dosed with 100 ng TC/cm2. Within 16 h, apical swelling of the columnar cells occurswith vesicle-like structures seen in the gut lumen. At 40 h, there is a complete dissolution of the larval gut lining and the presence of cellulardebris in the lumen. Bars � 50 �m.

TABLE 9. Chitinase assay

Chitinase Substrate

Activity (�mol of p-nitrophenol released after 30 min at 37°C)

TC (0.1 �g/ml) Standard chitinasea

(0.05 �g/ml) �TC strain

Exochitinase 4-Nitrophenyl-N-acetyl--D-glucosaminide 0.017 0.57 0.0034-Nitrophenyl-N-N�-diacetyl--D-chitobioside 0.0747 0.145 0.009

Endochitinase 4-Nitrophenyl-N-acetyl--D-N,N�,N�-triacetylchitotriose 0.1211 0.108 0.005

a Chitinase from Trichoderma viride (catalog no. CS0980; Sigma Aldrich).



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sedimentation of either Chi1, YenA1, or YenA2 was observedwith the Y. entomophaga MH96 chi2 mutant. SDS-PAGE ofthe ultracentrifuged pellets of Y. entomophaga MH96::9 (yenBmutant) showed that only the Chi1, Chi2, YenA1, and YenA2proteins pelleted. Combined, these data indicate that theseproteins form a subcomplex, similar to X. nematophila XptA1TCA, a tetramer-like structure described by Lee et al. (43), inwhich the TCB and TCC components are theorized to interact.Further credence to the formation of a Chi1, Chi2, YenA1,and YenA2 complex is their juxtaposed gene locations, of chi1and chi2 to yenA1 and yenA2, a gene order similar to their P.luminescens TTO1 orthologues (plu2461-plu2458) (Fig. 1C).

Western immunoblot and LC-ESI-MS/MS analysis identi-fied several low-molecular-weight YenA1 and YenA2 bandsindicative of proteolytic cleavage. N-terminal sequencing anal-ysis identified YenA2 cleavage products that correlated withthe relative positions of the predicted protease cleavage sites ofthe P. luminescens TC toxins TcaB, TcbA, and TcdA (5) (Table8; see Fig. S3 in the supplemental material). The reason behindthis cleavage is unknown but may relate to a bacterium-derivedprotease. N-terminal sequence analysis also identified the pre-dicted signal sequence cleavage site of the OmpA and OmpCproteins, which are the main components of outer membranevesicles (OMVs) (48). An additional protein that copurifiedwith the TC was the GroEL protein, a bacterial chaperoninthat facilitates the folding and transport of proteins (13, 57)and is often associated with OMVs (37). It is of interest thatthe �TC strain contained proteins with masses similar to thoseof Chi1 and Chi2 and an additional unidentified band locatedat �100 kDa (Fig. 4A, UC1P). This may indicate that in Y.entomophaga MH96, the TC predominantly associates withOMVs and that in the TC deletion variant other proteins withless affinity to OMVs are able to associate with the OMVs dueto nonsteric hindrance by the TC.

Using C-terminal glycogen synthase kinase (GSK) epitopetags fused to the N-terminal amino acid residues of the Y. pestisKIM TC-like protein’s TCA (YitA and YitB), TCB (YitC),and TCC (YipA and YipB), Gendlina et al. (27) identified thatthe GSK fusions are released by the Y. pestis type III system.The authors also found that under conditions optimal for typeIII secretion in Y. pseudotuberculosis, YitB could be detected inthe culture supernatant. Bioassay analysis of sonicated filtratesderived from Y. entomophaga MH96 cultures grown at 25, 30,or 37°C showed that they were still active on insects, suggestinga temperature-dependent release of the TC from the cell.Electron microscopy identified OMVs in the ultracentrifugedpellet, and scanning electron microscopy showed large blebsresembling OMVs on the outer cell wall of Y. entomophagaMH96 (Fig. 6B). OMVs have been shown to be involved in thetransport of degradative compounds, DNA (19), and toxins(39, 40), out of the bacteria. Along with the predicted chaper-one function of the GroEL protein, it is tempting to speculatethat GroEL and OMVs, respectively, are important in theassembly and transport of the TC out of Y. entomophagaMH96.

Through electron microscopy, pyramid-like structures wereobserved (Fig. 6A), and these structures were not seen in the�TC deletion variant. Given the size and purity of the fractionsas assessed by SDS-PAGE, the pyramid-like structures arelikely to be the TC. The semipurified TC was found to have

high endochitinase activity and may therefore have an impor-tant role in the degradation of insect-based chitin such as thatfound in the peritrophic membrane (44). Regev et al. (58)demonstrated that the S. marcesens-derived ChiAII endochiti-nase is able to cause mortality alone and can act synergisticallyto enhance the Bacillus thuringiensis K26-21 �-endotoxin crys-tal CryIC protein activity toward neonate Spodoptera littoralis.The estimated size of the X. nematophila TCA component(XptA1) is 16 by 19 nm (43), and the peritrophic membranefiltration cutoff point for type I and II peritrophic membranesis between 2 and 36 nm (44). The TC-associated chitinases maytherefore enhance the activity of the core TC by facilitating thedegradation of the chitin components of the insect intestinaltract and or other components of the insect. The TC was alsoable to cause lethality when injected into the larval hemocoeliccavity of G. mellonella, suggesting that the TC interacts with ageneric conserved receptor, a scenario postulated by Bowen etal. (5), who identified both oral and hemocoel-based M. sextainsecticidal activity with the P. luminescens toxin complex(TCA).

Histological examination of P. xylostella after the larvae hadbeen fed purified TC showed that within 48 h after ingestion,there was a general dissolution of the gut with expansion anddislodgement of the columnar cells (Fig. 7). Blackburn et al.(2) observed a similar phenomenon with P. luminescens TCAtoxin toward the gut of M. sexta and drew parallels to thehistological effects of the B. thuringiensis � endotoxins, vegeta-tive insecticidal protein Vip3A, and cholesterol oxidases.

Analysis of the PAIYe96 DNA sequence identified two 76-bptRNAGly DNA sequences and a G�C content that were dif-ferent from those of the bacterial genome. These are traits thattypify a pathogenicity island (26, 29). Within PAIYe96, theG�C content of the DNA encompassing the TC gene clusterlocated 5� of the remnant methyltransferase is 44.5%, while theregion located 3� encompassing yenUVTW is 40.1%, indicatingthat these regions may have been acquired at a different timepoints. Bioassay of the �U-W strain toward C. zealandica lar-vae showed no alteration in the ability to cause disease, sug-gesting that the yenUVTW genes may have some other role inthe ecology of Y. entomophaga MH96. The overall G�C con-tent of PAIYe96 (43.8%) is more similar to the genomic G�Ccontent of P. luminescens subsp. laumondii TTO1 (42.8%),Photorhabdus asymbiotica (42.2%), and X. nematophilus (45%)than to Yersinia species (Y. pestis and Y. pseudotuberculosis,47.6%) and Y. enterocolitica (47.3%). Phylogenetic analysis ofYenA2 showed that its closest relatives were the TCA proteins,TccB2 (Plu2459) of P. luminescens subsp. laumondii TTO1(22) and the X. nematophilus protein XptD1 (50). The targetspecies of either of these proteins has yet to be determined.The YenA2 protein was phylogenetically distinct from otherYersinia species (Fig. 2). DNA comparison of the Y. ento-mophaga MH96 chi1 and chi2 genes identified 67 to 70% DNAidentity to the chitinase genes of P. luminescens subsp. laumon-dii TTO1 (Fig. 1C) and the chi gene of X. nematophilus that islocated juxtaposed to the TCA-like genes xptD1 and xptAI (50).This suggests that the TC gene cluster may have been acquiredby horizontal gene transfer from one of these species. Theabsence of functional transposase, integrase, or other mobilitygenes within or flanking PAIYe96 indicates that the PAI is



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nonmobile and that PAIYe96 may therefore be in the finalstages of integration with the host genome.

We have provided the first evidence of an orally active TCderivative from a Yersinia species. Its broad range of activityagainst a number of insect pest species indicates that it mayprove useful as an alternative to B. thuringiensis toxins for usein insect control (7). To date, attempts to define parameterswhere toxin is not produced have proved unsuccessful (Hurst,unpublished). The ability to rapidly purify and visualize the TCwill allow a detailed protein structural analysis and facilitatestudies defining the TC insect-associated receptor.

ACKNOWLEDGMENTS

We are grateful to Richard Townsend for the collection of larvae ofthe grass grub (C. zealandica), redheaded cockchafer (A. couloni),Tasmanian grass grub (A. tasmania) and chafer beetles, Odontria sp.,and the rearing and provision of the white diamondback moth (P.xylostella). We thank Anette Becher for YenA2 phylogenetic anal-ysis and Michael Landsberg of the Institute for Molecular Biosci-ence, University of Queensland for proofreading the manuscript.

This study was supported by contract C10X0804 from the NewZealand Foundation for Research, Science and Technology. We haveno conflicts of interest.

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