Construction of a bioinsecticidal rhizosphere isolate of<i>Pseudomonas fluorescens</i>

Abstract

The cryIVB gene from Bacillus thuringiensis morrisoni PG-14 was cloned and expressed in Escherichia coli. A gene cassette was constructed that placed the gene under the control of the tac promotor. Three Pseudomonas ‘suicide’ vectors were made by cloning chromosomal DNA fragments from the root-colonizing Pseudomonas fluorescens strain P1 into plasmid pSUP202. The kanamycin resistance gene nptII and the cryIVB gene cassette were cloned within the Pseudomonas sequences. These constructs were introduced into the root-colonizing strain Pseudomonas fluorescens P1. Southern blot hybridizations demonstrated that the nptII and cryIVB genes were integrated into the chromosome whereas vector sequences were not. Expression of the cryIVB protein by transgenic Pseudomonas cells was demonstrated by Western blot analysis. Cell cultures of the transformed P. fluorescens were found to be toxic towards larvae of the malaria mosquito Anopheles stephensi and to leatherjackets, the larvae of Tipula oleracea.