Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame ( cyt1Ca ) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Abstract

Insecticidal crystal proteins of. This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3 end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.

Cyt1Aa Toxin: Crystal Structure Reveals Implications for Its Membrane-Perforating Function

“…Two internal salt bridges between residues R78-E196 and K163-D72 are designated by blue broken lines. Charged residues K154 and E164, which have been identified as important for Cyt1Aa toxicity 29 and are not conserved in Cyt1Ca, are designated by sticks and labeled in red (D240 is not shown, as there is no observed electron density for it). (b) The α/β architecture of the Cyt1Aa monomer, characterized by a central β-sheet (yellow) surrounded by two outer α-helical layers (red).…”

Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

“…Production of Cyt1Ca showed no such effects (data not shown), indicating that this protein is not toxic either to mosquito larvae or to E. coli cells, although Cyt1Ca mutants have been produced that do display effects on the host bacterium, but not on mosquitoes (Itsko et al, 2005).…”

“…2). Cyt1Ca-producing cells, however, were unable to supplement the activity of Cry4Aa, even at a ratio of Cyt1Ca : Cry4Aa as high as 4 : 1 (Itsko et al, 2005).…”

“…A new set of bioassays was performed with the truncated clones using fourth-instar mosquito larvae (Fig. 6B), but no significant differences in mortality (Itsko et al, 2005) or synergism with cry4Aa-expressing cells were observed with the truncated Cyt1Ca.…”

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

“…Although further studies are required to elucidate the precise mechanism of its stabilizing effect on the parasporal composite, the presence of the highly conserved metallophosphatase (MPP) and ricin-like beta-trefoil fold domains is suggestive of its function. In this regard, the structural and functional features of the carbohydrate-binding activity of the ricin-like beta-trefoil fold have been well characterized in proteins of eukaryotic and prokaryotic origin, including plant ricin, the serotype A hemagglutinating protein (HA33/A) of the botulinum neurotoxin of Clostridium botulinum, a few Cry and Cyt proteins of B. thuringiensis, and Mtx toxins of Bacillus sphaericus (1,6,7,12,13,15,28). Thus, Bt152 could be a novel lectin that putatively interacts with carbohydrate moieties of the multilamellar fibrous matrix and/or in Cry11Aa, Cy4Ba, and Cyt1Aa.…”