|Williamson, David - STATE UNIV NY|
|Rottem, Shlomo - HEBREW UNIVERSITY|
|Klein, Meir - ARO VOLCANI CENTER|
Submitted to: Oxygenic Photosynthesis: The Light Reactions
Publication Type: Other
Publication Acceptance Date: October 1, 1995
Publication Date: N/A
Interpretive Summary: Toward development of spiroplasmas (helical cell wall-less bacteria) as novel toxin-delivery systems for biocontrol of beetle pests in the United States (Colorado potato beetle) and Israel, media for culturing beetle-associated spiroplasmas were improved. Surveys of these spiroplasmas were conducted to provide strains for genetic engineering. Genetic material isolated from these spiroplasmas was used to construct a vector (vehicle) for transferring genes into spiroplasmas. Means for moving this vector into spiroplasmas were also optimized. With completion of the vector and the means for delivering it, we are poised to genetically engineer spiroplasmas with genes that will express toxins lethal to our target beetles, thus providing an effective and inexpensive alternative to conventional means of beetle control. This research will be of interest to scientists attempting to genetically engineer bacteria for pest control. Eventually, this technology would be transferred to biocontrol pest management specialists. This work may also be of use to clinicians developing better means for cultivating mycoplasma diseases associated with veterinary and human diseases.
Technical Abstract: Toward development of spiroplasmas as novel toxin-delivery systems for biocontrol of beetle pests in the United States (Leptinotarsa decemlineata) and Israel (Maladera matrida), media for cultivating beetle-associated spiroplasmas were improved and surveys of these spiroplasmas were conducted to provide transformable strains. Extensive surveys of spiroplasmas yielded promising extrachromosomal elements for vector constructs. One, plasmid pCT-1, was cloned, characterized, and used as a source of spiroplasma origin of replication in our shuttle vectors. The fibrillin gene was isolated and sequenced and its strong promoter was also used in the constructs. Means for transforming these vectors into spiroplasmas were developed and optimized, with electroporation found to be suitable for most applications. Development and optimization of means for using large unilamellar vesicles (LUVs) in spiroplasma transformation represents a breakthrough that should facilitate insertion of large clusters of virulence genes. With completion of the vector, we should thus be poised to genetically engineer spiroplasmas with genes that will express toxins lethal to our target beetles, thus providing an effective and inexpensive alternative to conventional means of beetle control.