Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads
- 1 November 1986
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 168 (2) , 982-989
- https://doi.org/10.1128/jb.168.2.982-989.1986
Abstract
Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).This publication has 18 references indexed in Scilit:
- The transposon Tn5 carries a bleomycin-resistance determinantGene, 1984
- Role of the IS50R proteins in the promotion and control of Tn5 transpositionJournal of Molecular Biology, 1984
- Fate of the insecticidal, proteinaceous parasporal crystal of Bacillus thuringiensis in soilSoil Biology and Biochemistry, 1984
- A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative BacteriaBio/Technology, 1983
- Tn5 transposition and its regulationCell, 1982
- The entomocidal toxins of Bacillus thuringiensisPharmacology & Therapeutics, 1981
- The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomologyCell, 1981
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Isolation of metabolic plasmid DNA from Pseudomonas putidaBiochemical and Biophysical Research Communications, 1977
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970