Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus.

Abstract

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...

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

“…To determine the location of the cryIF gene in B. thuringiensis EG6345 and EG6346 and to compare its location with that of the cryIA(b) gene present within strain EG6345, plasmid DNAs from EG6345 and EG6346 were resolved by agarose gel electrophoresis according to the method of Gonzalez et al (14) (Fig. SA).…”

“…aizawai EG6345 was isolated from a maize grain dust sample by using previously described procedures (11). Plasmid profiles were determined for each strain by electrophoresis through agarose gels (14). The crystal-negative (Cry-) strain B. thuringiensis HD73-26, which is a cured derivative of HD-73 containing a single 4.9-MDa plasmid, was used as a recipient for transformation of recombinant DNA constructs into B. thuringiensis (11, 13a (31).…”

“…The genes encoding ICPs have been localized to large (>30-MDa) plasmids (14,15), and various ICP genes have been cloned and characterized (for a review, see reference 20). Generally, the sequences of genes encoding proteins active on different orders of insects are not well conserved.…”

A Brazilian Bacillus thuringiensis strain highly active to sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

“…The extraordinary diversity of B. thuringiensis strains and toxins is, at least in part, due to a high degree of genetic plasticity. Most cry genes, which code for these toxins, reside on large (>30 MDa) plasmids (10,22,25) that can harbour one or several delta-endotoxin genes, with some being conjugative in nature (23). This plasticity is further enhanced by mobile elements, often found flanking cry genes (27).…”

Plasmids and Insecticidal Activity of Delta-endotoxin Crystals fromBacillus thuringiensisvar.israelensis

“…When a certain l'lasmid was transferred into an acrystalliferous strain of B. thuringiensis or Bacillus cereus, these strains were transformed into crystal-forming strains. 8 ) The delta-endotoxin protein genes from four strains, B. thuringiensis var. kurstaki HD I-Dipel,9) kurstaki,10) berliner 1715 11 ) and kurstaki HD73,12) carrying strong insecticidal activity against lepidopterous insects, were cloned in a strain of Escherichia coli.…”