Construction ofBacillus thuringiensiswild-type S76 and Cry–derivatives expressing a green fluorescent protein: two potential marker organisms to study bacteria–plant interactions

Collectively, the species Bacillus thuringiensis , Bacillus cereus , and Bacillus anthracis represent microorganisms of high economic, medical, and biodefense importance. Although the genetic correlation and pathogenic characteristics have been extensively dissected, the ecological properties of these three species in their natural environments remain poorly understood. Thus, a tractable marker for detecting these bacteria under specific environmental and physiological conditions is a valuable tool. With this purpose, a plasmid (pAD43-25) carrying a functional gfp gene sequence (gfpmut3A) was introduced into the wild-type strain Bacillus thuringiensis subsp. kurstaki S76, which bears approximately 11 plasmids, allowing constitutive synthesis of green fluorescent protein (GFP) during vegetative growth (strain S76GFP+). Additionally, this vector was transferred to a plasmid-cured (Cry–) B. thuringiensis host. Bright green cells were detected by fluorescence microscopy in both recombinants by 2 h after inoculation in liquid medium and could be seen throughout the remaining cultivation time until complete sporulation was accomplished. For strain S76GFP+protein profile and plasmid DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. Thus, in addition to the potential of strain S76GFP+as a marker organism in bacteria–plant interaction studies, the production and stability of active GFPmut3a make this unique expression system a useful experimental model to study adaptive changes of host–plasmid as well as plasmid–plasmid relationships in a population of cells stressed by the production of a recombinant protein.