Integrative Cloning, Expression, andStability ofthecryl4 (c) GenefromBacillus thuringiensis subsp. kurstaki ina Recombinant Strain ofClavibacter xyli subsp. cynodontis

A bacterial endophyte was engineered forinsecticidal activity against theEuropean corn borer. ThecrylA(c) gene fromBacillus thuringiensis subsp. kurstaki was introduced intothechromosome ofClavibacter xyli subsp. cynodontis byusing an integrative plasmid vector. Theintegration vectors pCG740andpCG741included the replicon pGEM5Zf(+), whichismaintained inEscherichia coli butnotinC.xyli subsp. cynodontis; tetMas a markerforselection inC.xyli subsp. cynodontis; andachromosomal fragment ofC.xyli subsp. cynodontis toallow forhomologous recombination between thevector andthebacterial chromosome. Insertion ofvector DNA into thechromosome was demonstrated byDNA hybridization. Recombinant strains MDR1.583andMDR1.586 containing thecryL4(c) genewere showntoproduce the133,000-kDa protoxin andseveral smaller immunoreactive proteins. Bothstrains wereequally toxic toinsect larvae inbioassays. Significant insecticidal activity was demonstrated inplanta. ThecryL4(c) geneandthetetM geneintroduced intostrain MDR1.586 were shownto bedeleted fromsome cells, thereby giving rise toa noninsecticidal segregant population. InDNAhybridization experiments andinsect bioassays, these segregants were indistinguishable