The recently discovered high-frequency transfer of plasmids between strains of Bacillus thuringiensis was used to study the genetic relationship between plasmids and production of the insecticidal 8-endotoxin crystal. Three strains of B. thuringiensis transmitted the Cry' (crystal-producing) phenotype to Cry-(acrystalliferous) B. thuringiensi recipients. Agarose gel electrophoresis showed that one specific plasmid from each donor strain was always present in Cry' "transcipients." The size of the transmissible crystal-coding plasmid varied with the donor strain, being 75 MDal (megadaltons) in size in HD-2, 50 MDal in HD-73, and 44 MDal in HD-263. Immunological analysis showed the Cry' transcipients to be hybrid strains, having flagella of the recipient serotype and crystals of the donor serotype. These results demonstrate that the structural genes for the 6-endotoxin are plasmid borne. Crystal-coding plasmids also transferred into two strains ofthe related species Bacillus cereus and yielded transcipients that produced crystals of the same antigenicity as the donor strain.The Gram-positive sporulating bacterium Bacillus thuringiensis is ofspecial scientific and economic interest because it produces an insecticidal toxin (known as the &.endotoxin) lethal to larvae of a wide range of lepidopterans as well as some dipterans (1). S-Endotoxin appears during sporulation as a crystalline inclusion (the parasporal crystal) that is phase-refractile, proteinaceous, and bipyramidal in most strains (2). Natural isolates of B. thuringiensis have been classified into at least 19 varieties on the basis oftheir flagellar and crystal antigens and their spectra of insecticidal activity (1, 3, 4). The closely related species B. cereus is distinguished by inability to produce the 8-endotoxin (1).Several inconclusive studies have attempted to determine whether the toxin genes are located on extrachromosomal plasmids, which are apparently ubiquitous in strains of B. thuringiensis (5, 6). We have recently implicated specific plasmids in the production of 8-endotoxin by several strains of B. thuringiensis, through analysis ofmutants cured ofindividual plasmids (7). These curing studies could not show whether the plasmids carried the actual toxin gene(s) or, possibly, regulatory genes controlling the expression ofchromosomal toxin genes. A means of genetic transfer was needed to show how a crystal-coding plasmid affected the phenotype of an acrystalliferous (Cry-) strain.We recently discovered that certain B. thuringiensis plasmids could be transmitted between two strains during growth in mixed culture, at frequencies of up to 75% of recipient colonies examined (8). Preliminary results showed that one plasmid previously implicated in toxin production could transfer into a Cry-strain ofa different flagellar serotype, yielding crystalliferous (Cry') transcipients (by "transcipients" we mean recipient cells that have acquired one or more plasmids from a donor strain*). These transcipients, though Cry', still exhibited the basic pla...

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.