THE PROTOXIN COMPOSITION OF BACILLUS-THURINGIENSIS INSECTICIDAL INCLUSIONS AFFECTS SOLUBILITY AND TOXICITY

Most Bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid encoded F-endotoxin genes and package related protoxins into a single inclusion, It was previously found that in B. thuringiensis subsp, aizawai HD133, which produces an inclusion comprising the CryIAb, CryIC, and CryID protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mDa plasmid containing the cryIAb gene, As a result, inclusions produced by the cured strain were less readily solubilized at pH 9.2 or 9.5 and had a decreased toxicity for Plodia interpunctella, despite the presence of the CryIC protoxin, which was active when solubilized, These results suggested that protoxin composition was a factor in inclusion solubility and toxicity and that the cryIAb gene, which is also present on an unstable plasmid in several other subspecies, may have a unique role in inclusion solubility and toxicity, Introduction of a cloned copy of this gene into the plasmid-cured derivative of B. thuringiensis subsp. aizawai HD133 resulted in an increase in the solubility at pH 9.2 of all of the inclusion proteins from less than 20% to greater than 45% and a lowering of the 50% lethal concentration (LC(50), in micrograms [dry weight] per square centimeter) of inclusions for Spodoptera frugiperda from 35 to 10, These values are the same as those found with inclusions from B. thuringiensis subsp, aizawai ai HD133, and in all cases, the LC(50) of the solubilized protoxins was 10, Transformants containing related cryIA genes produced inclusions which were more than 95% solubilized at pH 9.2 but also had LC(50) of 10. The presence or absence of a particular CryIA protoxin is thus a major factor in inclusion solubility and toxicity, Since there is instability of a plasmid containing the cryIAb gene in many of these subspecies, the inclusions produced by a population of these cells must be heterogeneous in terms of solubility and thus toxicity. Such flexibility may be of adaptive value.