A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis.
Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.
Department of Entomology, University of California, Riverside, Riverside, California, USA.
Mercedes
M
Diaz-Mendoza
Dennis K
DK
Bideshi
Brian A
BA
Federici
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D008853:Microscopy; D008969:Molecular Sequence Data; D010957:Plasmids; D020543:Proteome / Q000032:analysis*; D013171:Spores, Bacterial / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D020543:Proteome