A 54-Kilodalton Protein Encoded by pBtoxis Is Required for Parasporal Body Structural Integrity in Bacillus thuringiensis subsp. israelensis

Abstract

cStrains 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.

Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis

“…One is a 54-kDa protein, containing metallophosphatase and ricin-like domains. Although this protein is not a toxin, it was shown to be specific to the parasporal body and important for its structural integrity (Diaz-Mendoza, Bideshi and Federici 2012 ). The other two are the accessory proteins P19 and P20, indispensable for correct folding of Cry and Cyt proteins (Manasherob et al.…”

Contributions of 5′-UTR and 3′-UTR cis elements to Cyt1Aa synthesis in Bacillus thuringiensis subsp. israelensis

“…israelensis to aggregate the four mosquitocidal proteins (Cyt1Aa, Cry4Aa, Cry4Ba, Cry11Aa) into a parasporal body delimited by fibrous matrix of unknown composition. This results in a highly effective PB capable of killing sensitive dipteran larvae, i.e., it is much more toxic per unit weight than any of the endotoxins alone (Diaz-Mendoza et al, 2012). These toxins function synergistically, with the primary synergist being Cyt1Aa, which by itself is not very toxic to mosquito larvae (Wirth et al, 1997).…”

“…Its lethality to mosquitoes, blackflies and midges is due to synergistic interactions among four major PB proteins, three Cry toxins (Cry11Aa, Cry4Aa, Cry4Ba) and the lipophilic Cyt1Aa cytolytic toxin, the primary synergist (Wu and Chang, 1985; Ibarra and Federici, 1986; Crickmore et al, 1995). Cyt1Aa constitutes ~55% of the PB, whereas Cry11Aa accounts for ~35%, and Cry4Aa and Cry4Ba together account for the remaining ~10% (Federici et al, 1990; Wu and Federici, 1993; Dervyn et al, 1995; Diaz-Mendoza et al, 2012). …”

Genetic diversity of Brazilian Bacillus thuringiensis isolates with toxicity against Aedes aegypti (Diptera: Culicidae)

“…Several subspecies of Bt have been reported to be highly toxic against dipteran insects, such as Bt israelensis (Bti), Bt entomocidus, Bt sotto, Bt jegathesan, Bt darmstadiensis, Bt medellin, Bt fukuokaensis, and Bt higo 40,5,14,60,29,49,41,24 . Bti is considered the most toxic to dipteran larvae 16,13,3 . The mosquitocidal activity of Bti is due to insecticidal proteins encoded by the cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba genes 4,3,55 .…”