APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1994, p. 501-5080099-2240/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Integrative Cloning, Expression, and Stability of the cryl4(c)Gene from Bacillus thuringiensis subsp. kurstaki in a

Recombinant Strain of Clavibacter xyli subsp. cynodontisJAY S. LAMPEL,It* GAYLE L. CANTER,2 MICHAEL B. DIMOCK,3 JEFFREY L. KELLY,'

JAMES J. ANDERSON,1t BRENDA B. URATANI,' JAMES S. FOULKE, JR.,' AND JOHN T. TURNER2

Department of Molecular Genetics,' Department of Microbial Ecology, 2 and Department of Entomology, 3Crop Genetics International, 10150 Old Columbia Road, Columbia, Maryland 21046

Received 18 August 1993/Accepted 18 November 1993

A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The crylA(c)gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp.cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included thereplicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a

marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allowfor homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA intothe chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586containing the cryL4(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunore-active proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity wasdemonstrated in planta. The cryL4(c) gene and the tetM gene introduced into strain MDR1.586 were shown tobe deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridizationexperiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall,these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control.

Clavibacter xyli subsp. cynodontis is a fastidious gram-posi-tive coryneform bacterium that naturally inhabits the xylem ofBermuda grass (Cynodon dactylon L.) (6). C. xyli subsp.cynodontis also colonizes the vascular system of corn (Zea maysL.) when artificially inoculated (16). High populations of C. xylisubsp. cynodontis are distributed in the xylem elements follow-ing the introduction of inoculum into the tissues of the whorlor stem of corn seedlings or after germination of corn seedinfused with the bacterium. C. xyli subsp. cynodontis does notenter developing kernels and is therefore not present in theprogeny seed of inoculated plants (5). Additionally, a DNAtransformation system and the development of plasmid cloningvectors have been recently reported (13, 19).

Introduction of genes coding for entomocidal proteins intoother host microbes has resulted in expression of larvicidalactivity for control of insect pests (14, 20, 25). Endophyticbacteria such as C. xyli subsp. cynodontis are potential candi-dates for systemic delivery of biopesticides within a host plantwithout direct manipulation of the plant genome. One targetfor this type of delivery system is the European corn borer(ECB) (Ostrinia nubilalis). This insect is widely distributed andis a significant pest throughout most of the U.S. corn belt (18).Although early-season foliar infestations are often controlledby application of chemical insecticides or commercial formu-lations of Bacillus thuringiensis to the whorl of corn, larvaeinfesting plants at or beyond pollen shed feed in exposed areas

only briefly before moving to the protection of the leaf sheath,from which they tend to burrow into the stalk (18).We have previously inserted a truncated form of the cryLA (c)

* Corresponding author. Phone: (410) 550-2919. Fax: (410) 550-2924.t Present address: Paragon Biotech, Inc., Hopkins-Bayview Alpha

Center, 5210 Eastern Ave., Baltimore, MD 21224.t Present address: General Medical Sciences, National Institutes of

Health, Bethesda, MD 20892.

gene from B. thuringiensis subsp. kurstaki HD73 fused with a

kanamycin resistance gene into the chromosome of C. xylisubsp. cynodontis by homologous recombination (23). Thisprototype strain (MDR1.3) exhibited activity against the ECBin diet assays but not in planta. On the basis of phenotypic lossof resistance markers in plating assays and DNA hybridizationanalysis, segregant bacterial cells lacking the entire integratedplasmid were detected in bermuda grass and in corn as early as8 weeks postinoculation, though they constituted less than 5%of the total endophytic population at that time. Presumedly,the gene loss was due to excision by recombination across theregion of homology since the DNA flanking the integratedplasmid was arranged as a direct repeat. It was predicted thatsegregation rates similar to that observed with strain MDR1.3would not result in significant loss of insecticidal activity incorn for improved strains. Moreover, using bermuda grass, thesegregation process was shown to function as a containmentfeature in that engineered genes were completely lost from theendophyte population over much longer periods of time. It iscrucial to determine the stability of new integration vectorscontaining the 8-endotoxin gene to ensure a desirable balancebetween insecticidal activity and persistence of the introducedgenes in the environment.To demonstrate the overall feasibility of controlling the ECB

in corn by using a genetically engineered endophyte, we haveintroduced into C. xyli subsp. cynodontis a version of thecrylA(c) gene that leads to increased levels of the 8-endotoxin.In this report, we describe the production of the 8-endotoxin,activity of a recombinant strain against the ECB, and the rateof segregation.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Bacte-rial strains and plasmids are given in Table 1. C. xyli subsp.

501

Vol. 60, No. 2

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

502 LAMPEL ET AL.

TABLE 1. Bacterial strains and plasmids

C. xyli subsp. cynodontisMDE1MDR1.583MDR1.586MDS1.1276MDS1.1277

E. coli DH5otF'

PlasmidspAM120pGEM5zf(+)pFL15pCG563pCG740

pCG741

Description'

Wild-type strain isolated from bermuda grass from eastern MarylandMDE1 with pCG740 as a chromosomal insertionMDE1 with pCG741 as a chromosomal insertiontet cryL4(c) segregant of MDR1.586tet cryL4(c) segregant of MDR1.586

F' +80d lacZAM15A (lacXYA-argF) U169 recAl endAl hsdRJ7 (rk-, Mk+) SupE44 A-thi-1 gyrA reLA

pCGL101 carrying EcoI F' tetM fragment (F::Tn9J6)Apr

pLP1201 carrying the cryL4(c) protoxin geneC. xyli subsp. cynodontis integration vector containing tetM; confers Tetr to MDE1C. xyli subsp. cynodontis integration vector containing tetM and the cryL4(c) gene

cassette inserted into pGEM5zf(+)C. xyli subsp. cynodontis integration vector containing tetM and the crylA(c) gene

cassette inserted into pGEM5zf(+) in the opposite orientation of pCG740

Source or reference

This workThis workThis workThis workThis work

BRL,* Gaithersburg,Md.

9Promega, Madison, Wis.1R. S. StearmanThis work

This work

a Ap, ampicillin; Tet, tetracycline; Bt, B. thuringiensis cryL4(c) gene; r, resistant.b BRL, Bethesda Research Laboratories.

cynodontis was grown as previously described (23). Escherichiacoli DH5otF' was used as the host for construction of the C. xylisubsp. cynodontis integration vectors. Culture conditions for E.coli were the same as those previously described (23), exceptthat 5 ,ug of tetracycline per ml was used in addition toampicillin when appropriate for selection of transformants.DNA manipulation and hybridization. High-molecular-

weight DNA was obtained from C. xyli subsp. cynodontis, usingprocedure I (lysozyme-sodium dodecyl sulfate [SDS] lysisfollowed by phenol-chloroform extractions and ethanol precip-itations) as described for Streptomyces total DNA isolation(11). Plasmid DNA isolated from E. coli was purified with CsCldensity gradients (17).DNA probes for hybridization analysis of C. xyli subsp.

cynodontis transformants and segregants were radioactivelylabeled with [ct-32P]dCTP (NEN Research Products, Boston,Mass.), using an oligolabeling kit (Pharmacia, Piscataway,N.J.). The PolarPlex Chemiluminescent Blotting Kit (Milli-pore, Bedford, Mass.) was used to make nonradioactiveprobes. For hybridizations, DNA was either transferred fromagarose gels to Magnagraph membranes (Schleicher &Schuell, Keene, N.H.) with a Vacublot apparatus (AmericanBionetics, Hayward, Calif.) or hybridized with probe directly indehydrated gels (15). The conditions for nonradioactive hy-bridizations and washes were as suggested by the manufacturer(Millipore). Hybridizing bands were visualized with Lumogen(Millipore). For radioactive probes, the hybridization bufferwas composed of 5 x SSC (1 x SSC is 0.15 M NaCl plus 0.015M sodium citrate), 0.1% SDS, 5 x Denhardt's solution (11),and 0.25 mg of denatured salmon sperm DNA (Sigma, St.Louis, Mo.) per ml. Posthybridization washes were the same asfor nonradioactive probes. Bands were visualized by autora-diography.

Transformation. Plasmid DNAs were introduced into C. xylisubsp. cynodontis by electrotransformation. Cells were grownin two 50-ml cultures of S-8 medium (6) supplemented with0.1% glycine and 0.2% bovine serum albumin to an opticaldensity at 600 nm of 0.186 to 0.206 and harvested by centrif-ugation at 8,000 x g. The pellets were washed twice with cold(4°C) distilled water and then once with cold 10.3% sucrose.The final pellet was resuspended in 400 ,ul of 10.3% cold

sucrose per 50 ml of culture. Approximately 5 x 109 CFU (50,ul of cells) were used for each transformation. To achieve themaximum field strength employing the Bio-Rad Gene Pulser(Bio-Rad, Richmond, Calif.), 0.2-cm cuvettes were used withsettings of 100 fl, 25 ,uF, and 2.5 kV. Immediately after voltageapplication, the cells were diluted in 400 ,ul of cold S27 broth(23), spread onto a 0.2-p.m cellulose acetate membrane (MicroFiltration Systems, Dublin, Calif.), and placed on the agarsurface of SC plates (23). After 16 h of incubation at 30°C, themembranes were transferred to SC plates containing 2 p.g oftetracycline per ml. The plates were then incubated for 7 to 10days before individual transformants appeared.

Immunodetection of the 8-endotoxin. Polyclonal antibodieswere raised in goats by subcutaneous injection of purifiedcrystals prepared from B. thuringiensis subsp. kurstaki HD-73.The crystals were solubilized in ammonia-1% 2-mercaptoetha-nol and lyophilized, and 1-mg portions were injected inFreund's incomplete adjuvant. High-titer antisera werepooled, the immunoglobulin G was purified by dialysis againstacetic acid (pH 3.5), and specific antisera were prepared byaffinity purification against immobilized denatured crystal pro-tein.The presence of the 8-endotoxin was detected in crude

extracts of C. xyli subsp. cynodontis by a Western blot (immu-noblot) technique (21). Total protein extracts of C. xyli subsp.cynodontis were prepared by using the Mini-Beadbeater (Bio-spec Products, Bartlesville, Okla.) with 0.1-mm-diameter zir-conium beads (Biospec). Cellular proteins were solubilized byboiling in sample buffer (2% SDS, 1% 2-mercaptoethanol, 0.1M Tris-HCl [pH 6.8]) and separated by electrophoresis on12.0% SDS-12% polyacrylamide gels.The gels were electrophoretically blotted onto Immobilon P

membranes (Millipore). The membranes were blocked with1% bovine serum albumin, treated with affinity-purified anti-B.thuringiensis 8-endotoxin antibody, and visualized by using analkaline phosphatase-conjugated second antibody (Pierce,Rockford, Ill.) and the nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate toluidinium color reagent kit (KPLLaboratories, Gaithersburg, Md.). Sizes of immune stainedbands were determined with prestained molecular weightstandards (Amersham Inc., Arlington Heights, Ill.).

Strain or plasmid

APPL. ENVIRON. MICROBIOL.

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

crylM(c) EXPRESSION IN C. XYLI 503

Insect bioassay. Cell mass was harvested from 7-day-oldstreak cultures on SC plates, suspended in phosphate-bufferedsaline (PBS) to a standard optical density (y = 420 nm), anddiluted serially in PBS to the desired concentrations forbioassay. Each dilution was used to treat an agar-based wheatgerm-casein insect diet (BioServ, Frenchtown, N.J.) in 128-wellpolystyrene bioassay trays (C-D International, Pittman, N.J.)by pipetting 50 ,ul of the proper dilution onto the diet surfacein each of five replicate wells. Control wells contained diettreated with sterile PBS. Trays were agitated gently to wet theentire surface of the diet in each well and were then placedunder a sterile hood until the PBS had either dried or soakedinto the diet surface. Ten neonate ECB larvae were placed ineach well; then the trays were sealed with ventilated adhesivecovers and incubated at 27°C under a photoperiod of 16:8 h(light/dark). Larval mortality was scored after 4 days. Two setsof bioassays were conducted. Bioassay A included strainsMDR1.3, MDR1.586, and MDE1 and was conducted onlyonce. Bioassay B compared MDR1.586, MDR1.583, andMDE1 and was conducted in triplicate, and the resultingmortality data were subjected to probit analysis (7), using thePOLO-PC program (12).

In planta activity. Field corn (Z. mays) seeds were planted in3.8-liter pots containing a standard soil mix (Metro-Mix; GraceSierra, Milpitas, Calif.) and grown in a greenhouse. Seedlingswere inoculated with C. xyli subsp. cynodontis recombinantstrain MDR1.3 or MDR1.586 or wild-type isolate MDE1 byinjecting approximately 107 CFU into the meristem area 1 cmabove the soil line 4 weeks after planting. Control plants weresham inoculated with sterile PBS. Colonization was confirmed4 weeks after inoculation by microscopic detection of C. xylisubsp. cynodontis in sap expressed from the midrib of one leafper plant, taken from a leaf midway up the plant that did notshow an inoculation scar. Noncontrol plants that could not beconfirmed as colonized were discarded from the study. Re-maining plants were arranged in a randomized complete blockdesign with eight replications, each containing six plants perinoculation treatment. A sample of eight plants per treatment(one per replicate) was sacrificed to provide stem sections fordetermination of endophyte titer by standard quantitativedilution plating methods (see description under segregationstudies below). The rest were infested at or near pollen shedstage with ECB by placing 30 neonate larvae on each plant witha camel hair brush, distributing them uniformly among five orsix leaf axils surrounding the primary ear. Each plant wasdissected 3 weeks after infestation to expose feeding cavitiesresulting from larvae tunneling in the stalk and ear shank. Thenumber of surviving larvae, feeding cavities, and sum of thelengths of all tunnels (to the nearest 0.5 cm) were recorded foreach plant.

Segregation analysis. The occurrence of segregant colonieshaving lost the introduced genes has been studied with recom-binant bacterial strains in both corn and bermuda grass. Themethods for the culture, inoculation, sampling, and detectionof recombinant bacterial strains in greenhouse-grown bermudagrass plants have been described previously (23). Eighteenbermuda grass plants were sampled at 6, 11, 18, and 25 weeksafter inoculation. One hundred colonies isolated from each ofthe 18 plants at each sample time were scored for growth onplates containing tetracycline. Segregation was also studied byusing field-grown corn (proprietary field corn hybrid) in thesummer of 1991. Recombinant strain MDR1.586 was added asa seed treatment prior to planting. The percentage of pheno-typic segregants was determined at four times during thegrowing season, using large numbers of colonies isolated frommultiple plants in replicated trials in Maryland (CGI Research

Farm, Ingleside) and Nebraska (NC+ Research Station, Hast-ings). General methodology for seed inoculation, sample prep-aration, phenotype assay, and DNA-DNA hybridization hasbeen described (23) and, as with bermuda grass, involvesscoring for growth on plates containing tetracycline. Followingphenotype determination, 1,084 Tets colonies (924 from cornand 160 from bermuda grass) and 86 Tetr colonies (75 fromcorn and 11 from bermuda grass) were analyzed, using DNA-DNA hybridization with nonradioactive probes. A greaternumber of Tet' colonies was chosen because this class in-creased and eventually predominated in plants. Specifically,dot blots of the various colony isolates were probed with thecryLA (c) sequence of B. thuringiensis and the tet sequence fromTn916.

In order to further characterize the segregants, two seg-regant cultures were arbitrarily chosen for in vitro growth ratecomparisons with strains MDR1.586 and MDE1. Methods forthe growth rate determination have been described previously(23).

RESULTS

Construction of pCG741. To increase expression of thecryL4(c) gene in C. xyli subsp. cynodontis over previous con-structions, a promoterless version of the gene was cloned intoan integrative promoter-cloning plasmid vector. The integra-tion plasmid pCG741 contains (i) pGEM5Zf(+), a repliconthat functions in E. coli but not in C. xyli subsp. cynodontis,thereby providing selection for integration into the C. xylisubsp. cynodontis chromosome; (ii) tetM, a tetracycline resis-tance gene from Tn916 (3) that is selectable in C. xyli; (iii) thecryL4(c) gene from B. thuringiensis subsp. kurstaki HD73 (2);and (iv) a chromosomal fragment (int2O9) from C. xyli subsp.cynodontis that provides a site for homologous recombination.This integration fragment was generated by cloning randomlydigested C. xyli subsp. cynodontis DNA into a pUC19 deriva-tive containing a tet gene. Introduction by transformation of alibrary of these fragments into C. xyli subsp. cynodontis led totetracycline-resistant transformants and the identification ofDNA fragments capable of mediating integration into thechromosome. One of these fragments was int2O9.The cryL4(c) gene was cloned as an NdeI fragment into

pGEM5Zf(+) (Fig. 1) so that the native B. thuringiensispromoters (1) were excised. Additionally, one of two stem-loopinverted-repeat structures (24) in the 3' flanking DNA wasremoved during this step. A synthetic oligomer containing aunique BamHI cloning site immediately upstream of thecryLA(c) gene and translation stop codons was then inserted atthe 5' end of the gene as shown in Fig. 1. The modified genewas moved as an ApaI cassette into pCG563, an integrationvector containing tetM and the chromosomal integration frag-ment, to make pCG740 and pCG741 (Fig. 1).

Structural analysis of pCG741 in C. xyli. subsp. cynodontis.pCG741 was introduced into strain MDE1 by electrotransfor-mation. Several Tetr transformants were analyzed for 8-endo-toxin production by Western blotting and insect toxicity (datashown below). One transformant was named strain MDR1.586and chosen for all further studies.The fate of pCG741 in C. xyli subsp. cynodontis was deter-

mined by DNA hybridization. To demonstrate that the plasmidhad integrated into the chromosome, genomic DNA wasisolated from strains MDE1 and MDR1.586 and analyzed byDNA hybridization. The DNAs were digested with Sfil, whichcleaves pCG741 once at a site located in the polylinker ofpGEM5Zf(+). The hybridization probe was int2O9. If pCG741was maintained as an extrachromosomal plasmid, then upon

VOL. 60, 1994

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

EcoRI.6SS /~Seal.332EtoRI,1381 Nde1,4077/

\ { ~~~~~~~~~~pGEMSZf(#)\ ~~~~~~~~~219S.Apal 3003 bps\Ndrel 2124,Ndef\ _ ~~~~~~2103.BstXI

SphlS67?3.Ndel Aps170.g ori/

// 1§\ ~~~~~~~Sca1,1210 BstXI,I

({ ~~~pCG737 89111|j till22\ / g : | g 11g~~~~~~~~~~~~I BamHI.41 BXIx60

\\ 5/ t~~~~~~~~~~~~olytinker

4071,EcoRlBstX~BstI.2987 BlI /6817.ApalNdef.3002 16805 SphlEcoRI,33SI 6746,14def

11982,BglII Sca1,1141 \} t112

\ jr~~~~AM pCG738

loos2Bglll~~~~~~~orBtQ 6831bps|209 pCG563 Or \Ndef.2933 \B

13007 bpg } \ i/%"-v_ _ \~~~~~~~~~~~~~Apol / 00EoI~'APll.2917o2s

Ndel,5475 MM&u1,292974Ndel.29iS

'EcoRI,332416196,Scal

IS044.ATJ1@.1S027,1BstElf- Ndef.937 |SS106A Ia Ndel,937

IS021.Barnli' I,/S027.TfailAsirrsi Ss23/\h "te Scaf.2936

861S.Bglll

8615.Bglll

504

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

cryL4A(c) EXPRESSION IN C. XYLI 505

1 2 3 1 2 3 4

kD13392

so

- 22.8 kb_-17.1 kb-16.2 kb

- 11.7 kb

FIG. 2. DNA structural analysis of C. xyli subsp. cynodontis trans-formed with pCG741 demonstrating chromosomal integration of theplasmid. All DNAs were digested with SfiI. Lanes: 1, MDE1; 2,MDR1.586; 3, pCG741. The blot was hybridized with the entireintegration sequence (int2O9).

Sfil digestion and hybridization, a 17.1-kb fragment corre-sponding to linearized plasmid would be detected. However, ifpCG741 was integrated into the C. xyli subsp. cynodontischromosome, the 17.1-kb fragment would disappear and twoother fragments would be generated since other Sfil sites arepresent in the genome bordering the plasmid insertion. Figure2 shows that when strain MDR1.586 DNA was digested withSfil and then hybridized to the probe, the hybridizing bandseen with wild-type MDE1 (lane 1) was replaced by two newfragments (22.8 and 11.7 kb). This indicates that pCG741 wasconverted to a linear form that was integrated into the C. xylisubsp. cynodontis chromosome at the site of homology corre-sponding to the integration sequence of the plasmid. Theminor band seen in lane 2 probably is incompletely digestedDNA. Further structural analysis of strain MDR1.586 by KpnIdigestion (which generates a circularly permitted map of theintegrated plasmid indistinguishable from pCG741) and hy-bridization with four different probes derived from pCG741indicated that no deletions or rearrangements had occurred(data not shown).Immunodetection of the 8-endotoxin. Protein extracts of C.

xyli subsp. cynodontis containing the cryLA(c) gene were pre-pared and analyzed by Western blot. As shown in Fig. 3,

FIG. 3. Expression of the B. thuringiensis subsp. kurstaki protoxingene [cryLA(c)] cloned in C. xyli subsp. cynodontis. Total proteinextracts of recombinant strains were separated through an SDS-12.5%polyacrylamide gel and transferred to an Immobilon P membrane. The8-endotoxin was visualized with polyclonal antisera directed againstcrystals of HD73. Lanes: 1, strain MDR1.586; 2, strain MDR1.583; 3,strain MDE1; 4, HD73 crystals (50 ng).

proteins immunoreactive to polyclonal antisera raised againstB. thuringiensis subsp. kurstaki HD73 crystals were present inMDR1.586. In addition to strain MDR1.586, strain MDR1.583was also tested for the production of the 8-endotoxin.MDR1.583(pCG740) differs from MDR1.586 only in that theorientation of the ApaI cassette containing the crylA(c) genecloned in the integrative plasmid is reversed from that inMDR1.586. Both recombinant strains produce the 133,000-kDa protoxin. In addition to the protoxin, there are severalother bands present. The origin of these smaller proteinspecies has not been determined. The 133,000-kDa bandrepresents approximately 10% of the total antigen presentdetermined by scanning densitometry (10). The production ofthe 8-endotoxin was unexpected since pCG740 and pCG741 donot contain any explicit promoter in front of the crylA4(c) gene.Attempts were made to increase the level of expression abovethat of MDR1.586 by placing several defined E. coli consensuspromoters (e.g., ptac) into the BamHI site of pCG741. Addi-tionally, a randomly generated genomic library of C. xyli subsp.cynodontis DNA was made by using xylE as a reporter gene.Potential promoters were screened in C. xyli subsp. cynodontis,and a few of these were cloned into BamHI of pCG741.Neither of these two approaches led to any significant increaseof insect toxicity.

Insect bioassay. In bioassay A, both C. xyli subsp. cynodontisMDR1.3 and MDR1.586 were toxic to ECB larvae, butMDR1.586 was considerably more so, causing greater than50% mortality even at the lowest concentration tested (Fig.4A). The mortality rate of larvae ingesting wild-type isolateMDE1 did not differ from that of PBS-treated controls (16%).

In bioassay B, both C. xyli subsp. cynodontis MDR1.586 andMDR1.583 were toxic to ECB larvae, but wild-type isolate

FIG. 1. Construction of integration vectors containing the crylA(c) gene from B. thuringiensis subsp. kurstaki. The cryL4(c) gene was initiallyisolated and subcloned from pFL15 (2). The NdeI fragment cloned into pGEM5Zf(+) does not contain the native Bacillus promoter. Afteraddition of the polylinker, the crylA (c) gene was moved as an ApaI cassette into the integration vector pCG563 to make pCG740 and pCG741.The DNA derived from C. xyli subsp. cynodontis that is required for integration is labeled int2O9.

VOL. 60, 1994

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

506 LAMPEL ET AL.

100 100

A /,_ . _~MDR1.586 B80 /80 .MDR.583

--MDE 1

60 6/ 01UMDR1.3

0.0 .01 01 1001 00 .

FIG. 4.Mraiyo- .nbllslra edn natfca itta

R40 MDR1.586 40

U1)on-u0 M DE1C) 20 20

o

0.001 0.01 0.1 0.001 0.01 0.1

Concentration (OD/mi)

FIG. 4. Mortality of 0. nubilalis larvae feeding on artificial diet that

was treated topically with different concentrations of C xyli subsp.

cynodontis strains in PBS. (A) Results of a single bioassay comparingMDR1.3, MDR1.586, and MDEL. (B) Results of a triplicate bioassay

including MDR1.583, MDR1.586, and MDEIL OD, optical density.

MDE1 did not cause mortality significantly greater than that of

the control group (14%) at any of the bioassay concentrations

(Fig. 4B3). The concentration-mortality curves of the two

recombinant strains were indistinguishable by probit analysis

(pooled 50% lethal concentration = 0.048 optical density [420nm] U per well; 95% confidence limits for 50% lethal concen-

tration = 0.040 to 0.056; slope ± standard error = 1.1680.069; goodness-of-fit, x2 = 3.823, df = 8).

In planta activity. Inoculation of corn plants with C. xylisubsp. cynodontis recombinant strain MDR1.586 resulted insignificantly lower ECB survival and less feeding damagecompared with plants inoculated with either PBS or wild-typeisolate MDE1. In contrast, larval survival and tunneling inplants inoculated with recombinant strain MDR1.3 were notsignificantly different from those of PBS or MDE1 controls butwere significantly greater than in plants inoculated withMDR1.586 (Table 2). The overall feeding damage (totalcentimeters of tunneling per plant) was reduced by about 60%in plants inoculated with MDR1.586 compared with PBS-inoculated controls.

Segregation analysis. The population of Tets cells in ber-muda grass increased rapidly after inoculation such that 41%of the colonies recovered were of the Tets phenotype after only6 weeks (Table 3). The proportion of Tets colonies continuedto increase, but the rate of increase slowed as the percentageapproached 100.

In field-grown corn plants, the increase in the proportion ofsegregants appears to be much slower. The percentage ofsegregants was 4.0 and 0.6 for Maryland and Nebraska,

TABLE 2. ECB larval survival and feeding damage in reproductivestage field corn colonized by different strains of C. xyli subsp.

cynodontis (or sham-inoculated with sterile PBS) in the greenhousea

C. xyli subsp. ECB tunnels per plant

Inoculation cynodontis Live ECBtreatment population per plant Total no. Total cm

(CFUIg)6

PBS 6.3 ± 0.3a 5.5 ± 0.3a 16.0 ± l.a

MDE1 8.47a 7.6 ± 0.3a 6.2 ± 0.3a 19.2 ± 1.2aMDR1.3 8.69a 7.0 ± 0.4a 6.2 ± 0.4a 17.4 ± 1.4aMDR1.586 9.07b 3.7 ± 0.3b 2.8 ± 0.2b 6.1 ± 0.6b

a Means within a column not followed by the same letter are significantlydifferent (Duncan-Waller k-ratio t test, a = 0.05).

In planta populations.

TABLE 3. Segregation of introduced genes in endophyticpopulations of bermuda grass previously inoculated with pure

cultures of recombinant strain MDR1.586

No. of antibiotic-Wk after sensitive % Antibiotic-sensitive

inoculation segregants/no. segregantsassayed'

6b 663/1,626 4111 1,332/1,800 7418 1,537/1,800 8525 1,601/1,800 89

a Total number assayed was 7,026.b Typically, 100 colonies per plant (18 plants) were tested for antibiotic

sensitivity. At 6 weeks postinoculation, fewer than 100 were recovered from someplants due to contamination, lower levels of colonization, etc.; hence, the numbertested was less than 1,800.

respectively, at the earliest sampling (June). The percentagesof segregants in these plants increased during the growingseason such that by the final sampling (August for Marylandand September for Nebraska) the percentages were 8.8 and 5.6for Maryland and Nebraska, respectively. All 86 Tetr coloniesrecovered from greenhouse-grown bermuda grass and field-grown corn were shown by DNA-DNA hybridization to haveretained both the tetM and the cryL4(c) genes. Conversely,those 1,084 colonies which were Tets did not hybridize to thetet or the cryL4(c) probe with the exception of 1 colony. Thiscolony, though Tets, gave a positive hybridization signal for thetetracycline marker. Analysis of this strain by Southern hybrid-ization and diagnostic PCR revealed a deletion within thetetracycline gene (data not shown).

Differences in in vitro growth rates were observed (Table 4).The recombinant strain MDR1.586 grew more slowly than thewild-type strain MDE1. The two segregant strains appeared tohave growth rates at least as fast as that of the wild type.

Structural analysis of tet cryL4(c) segregants of strainMDR1.586. In order to determine whether rearrangement ofthe adjoining chromosomal DNA occurred during the segre-gation process, genomic DNA was isolated from nine tetcryL4(c) segregants and digested with BglII. By using int2O9 asa probe and comparing the hybridization profile with that ofBglII-digested DNA from strains MDE1 and MDR1.586, thestructural integrity of the segregants was assessed.

Hybridization of the probe to BglII-digested pCG741 yieldedthree fragments of the expected sizes (1.0, 1.5, and 14.5 kb)(Fig. 5, lane 2). In addition to these fragments, two otherfragments (9.3 and 17.1 kb) were present from strainMDR1.586. These were derived from the chromosomal borderregions on each side of the plasmid insert. The sizes of thefragments were defined by the unmapped BglII sites containedin the adjacent chromosomal DNA.

TABLE 4. Comparison of growth rates of C. xyli subsp.cynodontis strains

Strain Specific growth Generationrate (h- I)a time (h)

MDE1 0.109 ± 0.003 6.4MDR1.586 0.094 ± 0.003 7.4MDS1.1292b 0.120 ± 0.003 5.8MDS1.1293b 0.110 ± 0.008 6.4

a Specific growth rates are averaged from four flasks. Means ± standarddeviations are given.

*tet crylA(c) segregant of MDR1.586.

APPL. ENVIRON. MICROBIOL.

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

cr-yIA (c) EXPRESSION IN C. AYLI 507

1234 5 6 78

14 5 kb-

9.3 kb -

:4Q

1.5 kb -

1.0 kb -

FIG. 5. DNA hybridization of BglII-digestelated from nine cryIA (c) tet segregants of strain:was the entire integration sequence (B and B':k DNA digested with HindIII; 2, pCG741 digestMDE1 DNA digested with BglII; 4, strain MDwith BglII; 5 to 13, segregant DNAs digested w

When BglII-digested DNA from strainonly the fragments derived from the bordand the 1.5-kb internal fragment were

pCG741 sequences were present exceptments (Fig. 5). All nine of the segregant-desame hybridization profile as that of strathat the excision of pCG741 from the (

reproducible and precise event. The ninwere then tested for activity against E(bioassays. None of the segregants exhitbackground levels (data not shown).

DISCUSSION

Insertion of plasmid DNA into the genccynodontis was accomplished by recombisequence homology between the plasmi(chromosome. Since single crossover eventgous recombination generate direct repea

9 10 1112 13 structure, excision by reversal of this reaction can lead to geneloss. It is critical that the integrated plasmid DNA in strainMDR1.586 propagated in plants be sufficiently stable to deliverthe 8-endotoxin to its intended target. If the segregation ratesin recombinant C. xyli subsp. cynodontis strains are too high,product performance during a growing season would be com-promised. On the other hand, if the segregation rates weresufficiently low, high populations of active bacteria would bemaintained for activity during a single growing season, but overlonger time periods the engineered genes would be eliminatedfrom the C. xyli subsp. cynodontis population if it becameestablished in the environment. Strain MDR1.586 appears to

yPs0* have a segregation rate in this desirable range, with an averageof only 7% of the colonies recovered from plants at the end ofthe season having lost the introduced genes. More stableconstructions could be made by inserting the cryIA(c) geneinto the chromosome by double reciprocal recombination. Inthis way, none of the integration vector DNA would be

* retained except the toxin gene. Such constructions mightprovide insect control similar to that of MDR1.586 but would

* pose additional environmental risks associated with prolongedgene retention.

After 25 weeks of growth in bermuda grass, 89% of theMDR1.586 cells tested had lost the tetM and the crylA(c)genes. The rate of gene loss from C. xyli subsp. cynodontis infield-grown corn appeared to be slower than that of green-house-grown bermuda grass. This may be due to fewer inplanta bacterial generations during the growing season of corn.Both the continuing genetic deletion events and the differentialgrowth rates of segregant and nonsegregant populations couldcontribute to the increase in the proportion of segregants in

,,* the C. xyli subsp. cynodontis population. We have previouslyshown that growth rate differences between a recombinantstrain of C. xyli subsp. cynodontis (strain MDR1.3) containingan integrated plasmid with multiple sites of homology to thechromosome and segregants of this strain were responsible for

* the increase of segregant populations (23). According to themathematical model describing instability of plasmid-bearing

d genomic DNA iso- microbes (4), the in vitro-determined growth rates of strainMDR1.586. The probe MDR1.586 and segregant strains were great enough to accountfrom Fig. 3). Lanes: 1, for the predominance of segregant populations. In plantated with Bglll; 3, straie growth rate studies must be done to determine whether this isiR1.586 DNA digeste actually the case for cells that have colonized bermuda grassuith Bglll. and corn.

Expression of the cryIA(c) gene was examined by determin-ing the activity of MDR1.3, MDR1.583, and MDR1.586

MDE1 was probed, against ECB in an artificial diet bioassay. All three recombi-er regions of int209 nant strains displayed activity as measured by mortality of ECBobserved since no compared with background levels for MDEI. Strain MDR1.3

the integration seg- appeared less toxic than MDR1.586, while MDR1.586 andrived DNAs had the MDR1.583 were equally toxic to larvae (Fig. 4).tin MDEI, showing The activity observed for both MDR1.583 and MDR1.586chromosome was a was unexpected since the crylA (c) gene cassettes cloned ine segregant strains pCG740 and pCG741 were oriented in opposite directionstB larvae in insect without containing any explicit promoters. In pCG740, the)ited toxicity above DNA adjacent to the cryIA(c) gene is derived from the

polylinker region of pGEM5Zf(+), whereas the DNA imme-diately upstream of the crylA(c) sequences in pCG741 isderived from the chromosome of C. xyli subsp. cynodontis.Preliminary SI mapping and primer extension experiments

Dme of C. xyli subsp. indicate that the polylinker region contains a fortuitous pro-Tnation mediated by moter that was introduced into these plasmids during theirand the bacterial construction (8). When defined E. coli-type promoters or

:s driven by homolo- potential Clavibacter promoters derived from a xylE screenits in the integrated were cloned into the BamHI site of pCG741, which places

1. .. I'll

4

., ; 6 .... .]A Oll'! . .Aft..

VOL. 60, 1994

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from

508 LAMPEL ET AL.

these promoters adjacent to the promoterless cryL4(c) gene,no increase in 8-endotoxin production was detected.

- In addition to the production of the 133,000-kDa protoxin,identical immunoreactive profiles were present in extractsisolated from both strains MDR1.583 and MDR1.586. Sincethe toxic form of CryIA(c) has an Mr of 68,000, it is not clearwhether the smaller material contributes to overall activity.

Results of the greenhouse in planta study demnonstrate thefeasibility of using an endophytic prokaryote, altered by thechromosomal insertion of an insecticide-coding gene, for con-

trolling an insect pest of a major agricultural crop. Underconditions simulating a moderately heavy infestation by ECB(six to seven live borers per plant; total tunnel length of 16 to19 cm in controls), inoculation of plants with C. xyli subsp.cynodontis recombinant strain MDR1.586 reduced overalldamage by about 60% (Table 2). Whether this level ofprotection is sufficient to provide economic control of ECB incommercial field corn must be determined in field trials inwhich the effect of ECB infestation on yields of inoculated anduninoculated corn plots can be assessed.

Previous work has shown the desirable ecological character-istics (e.g., plant dependency, lack of insect or seed transmis-sion, etc.) of wild-type and recombinant strains of C. xyli subsp.cynodontis (22). In this report, we show in planta biologicalactivity and suitable stability. Continuing work is aimed atconstructing strains expressing increased levels of the 8-endo-toxin that are compatible with current commercial corn hybrids.

ACKNOWLEDGMENTS

We acknowledge A. Aronson for supplying the cryL4(c) gene. Wealso thank Mary Greger and Kelly Smith for typing the manuscript.

REFERENCES

1. Adang, M. J., M. J. Staver, T. A. Rocheleau, J. Leighton, R. F.Barker, and D. V. Thompson. 1985. Characterized full-length andtruncated plasmid clones of the crystal protein of Bacillus thurin-giensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta.Gene 36:289-230.

2. Arvidson, H., P. E. Dunn, S. Strnad, and A. I. Aronson. 1989.Specificity of Bacillus thuringiensis for lepidopteran larvae: factorsinvolved in vivo and in the structure of a purified protoxin. Mol.Microbiol. 3:1533-1543.

3. Burdett, V., J. Iramine, and S. Rajagopalan. 1982. Heterogeneityof tetracycline-resistant determinants in streptococcus. J. Bacte-riol. 149:995-1004.

4. Cooper, N. S., M. E. Brown, and C. A. Caulcott. 1987. Amathematical method for analyzing plasmid stability in microor-ganisms. J. Gen. Microbiol. 133:1871-1880.

5. Crop Genetics International. 1988. EUP58788-EUP2: applicationfor an experimental use permit to ship and use a pesticide forexperimental purposes only. Document Control Office, Office ofPesticide Programs, U.S. Environmental Protection Agency,Washington, D.C.

6. Davis, M. J., A. G. Gillaspie, A. K. Vidaver, and R. W. Harris.1984. Clavibacter: a new genus containing some phytopathologeniccoryneform bacteria, including Clavibacterxyli sp. nov. subsp. nov.

and Clavibacterxyli subsp. cynodontis subsp. nov., pathogens thatcause ratoon stunting disease of sugarcane and bermudagrass

stunting disease. Int. J. Syst. Bacteriol. 34:107-117.

7. Finney, D. J. 1971. Probit analysis, 3rd ed. Cambridge UniversityPress, London.

8. Foulke, J. S., and S. Bartz. Unpublished data.9. Gawron-Burke, C., and D. B. Clewell. 1984. Regeneration of

insertionally inactivated streptococcal DNA fragments after exci-sion of transposon Tn916 in Escherichia coli: strategy for targetingand cloning genes from gram-positive bacteria. J. Bacteriol. 159:214-221.

10. Gruninger, R. H., J. T. Turner, A. Ricotta, and J. S. Lampel. 1991.Abstr. Annu. Meet. Soc. Ind. Microbiol., abstr. p38.

11. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton,H. M. Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H.Schremp£ 1985. Genetic manipulation of Streptomyces, a labora-tory manual. The John Innes Foundation, Norwich, United King-dom.

12. LeOra Software, Inc. 1987. POLO-PC: a users guide to probit orlogit analysis. LeOra Software, Berkeley, Calif.

12a.Lohnas, J. L. Unpublished results.13. Metzler, M. C., Y. P. Zhang, and T. A. Chen. 1992. Transformation

of the gram-positive bacterium Clavibacter xyli subsp. cynodontisby electroporation with plasmids from the IncP incompatibilitygroup. J. Bacteriol. 174:4500-4503.

14. Murphy, R. C., and S. E. Stevens, Jr. 1992. Cloning and expressionof the cryIUD gene of Bacillus thuringiensis subsp. israelensis in thecyanobacterium Aymenellum quadruplicatum PR-6 and its result-ing larvicidal activity. Appl. Environ. Microbiol. 58:1650-1655.

15. Purrello, M., and I. Balazs. 1983. Direct hybridization of labeledDNA to DNA in agarose gels. Anal. Biochem. 128:393-397.

16. Reeser, P. W., and S. J. Kostka. 1988. Abstr. Annu. Meet.Phytopathol. Soc., abstr. 223.

17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring Harbor Press,Cold Spring Harbor, N.Y.

18. Showers, W. B., J. F. Witkowski, C. E. Mason, D. D. Calvin, R. A.Higgins, and G. P. Dively. 1987. European corn borer: develop-ment and management. North Central Regional Extension Publi-cation No. 327. Iowa State University, Ames.

19. Taylor, J., R. S. Stearman, and B. B. Uratani. 1993. Developmentof a native plasmid as a cloning vector in Clavibacter xyli subsp.cynodontis. Plasmid 29:241-244.

20. Thanabalu, T., J. Hindley, S. Brenner, C. Oei, and C. Berry. 1992.Expression of the mosquitocidal toxins of Bacillus sphaericus andBacillus thuringiensis subsp. israelenis by recombinant Caulobactercrescentus, a vehicle for biological control of aquatic insect larvae.Appl. Environ. Microbiol. 58:905-910.

21. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

22. Turner, J. T., J. L. Kelly, and P. R. Carlson. Endophytes: analternative genome for crop improvement. In D. R. Buxton (ed.),International crop science I, in press. Crop Science Society ofAmerica, Madison, Wis.

23. Turner, J. T., J. S. Lampel, R. S. Stearman, G. W. Sundin, P.Gunyuzlu, and J. J. Anderson. 1991. Stability of the f-endotoxingene from Bacillus thuringiensis subsp. kurstaki in a recombinantstrain of Clavibacter xyli subsp. cynodontis. Appl. Environ. Micro-biol. 57:3522-3528.

24. Wong, H. C., and S. Chang. 1986. Identification of a positiveretroregulator that stabilizes mRNAs in bacteria. Proc. Natl.Acad. Sci. USA 83:3233-3237.

25. Zaritsky, A., V. Zalkinder, E. Ben-Dov, and Z. Barak. 1991.Bioencapsulation and delivery to mosquito larvae of Bacillusthuringiensis H14 toxicity by Tetrahymena pyriformis. J. Invert.Pathol. 58:455-457.

APPL. ENVIRON. MICROBIOL.

on August 9, 2019 by guest

http://aem.asm

.org/D

ownloaded from