Lb. Selinger et al., EXPRESSION OF A BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN GENE BY BACILLUS-PUMILUS, Canadian journal of microbiology, 44(3), 1998, pp. 259-269
The delta-endotoxin genes from Bacillus thuringiensis were introduced
into a rhizosphere-inhabiting Bacillus pumilus isolate to create a del
ta-endotoxin expression and delivery system for subterranean feeding i
nsects 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 pal
e western cutworm larvae. Three different cry genes from Bacillus thur
ingiensis subsp. kurstaki were cloned into high and low copy number ve
ctors and mated into Bacillus pumilus RB8. When carried on high copy n
umber vectors, cry genes appeared to inhibit sporulation and delta-end
otoxin 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 incl
usions. On low copy number vectors, the cry genes did not inhibit spor
ulation; however, production of delta-endotoxins was undetectable. Usi
ng a heat shock regime for enrichment of sporogenous crystalliferous v
ariants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy
number plasmid was obtained in which high level delta-endotoxin produc
tion occurred concomitant with sporulation. Synthesis of functional de
lta-endotoxin by this strain was confirmed by Western blot analysis an
d bioassay with pale western cutworm larvae. These results show that r
hizosphere-inhabiting bacilli are indeed a potential route for introdu
ction of delta-endotoxins to the root environment for biocontrol purpo
ses.