Expression of a Bacillus thuringiensis δ-endotoxin gene by Bacillus pumilus

Abstract

The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

The Bacillus subtilis Conjugative Transposon ICEBs1Mobilizes Plasmids Lacking Dedicated Mobilization Functions

“…2), that replicate by rolling circle replication (RCR) and are typically described as nonmobilizable (30,43,44,46), although very low efficiencies of mobilization of pC194 and a relative of pBS42 by the conjugative transposon Tn916 have been reported (38,45). The three plasmids used here contain an origin of replication but are not known to contain an origin of transfer or have mobilization functions.…”

Genetically Modified Bacillus thuringiensis Biopesticides

“…The highest transformation frequency of BMB171 with several common plasmids was 10 7 transformants/µg DNA; its transformation efficiency and expression properties are better than that of the recipient strains discussed (Li et al 2000;Li and Yu 1999). Moreover, in addition to Bt wild-type or acrystalliferous strains, various Bt ICP genes have also been successfully transferred into B. subtilis (Shivakumar et al 1986(Shivakumar et al , 1989Calogero et al 1989), Bacillus sphaericus (Poncet et al 1994(Poncet et al , 1997, Bacillus megaterium (Mettus and Macaluso 1990;Shivakumar et al 1989), and Bacillus pumilus (Selinger et al 1998), among others.…”

Genetic Improvement of Bt Strains and Development of Novel Biopesticides

“…(Udayasuriyan et al 1995), two bacteria that colonize the roots and leaves of many plants. Similarly, the cry genes from Bt were also introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delivery system control subterranean feeding insects such as Agrotis orthogonia (Selinger et al 1998). However, only poor levels of expression were achieved probably due to instability of introduced plasmids.…”