Bacillus thuringiensis and its pesticidal crystal proteins

Abstract

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

FIG. 1

Three-dimensional structures of Cry1A, Cry3A, and Cyt2A.

FIG. 2

Amino acid sequence blocks conserved among Cry proteins. For each block, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid (indicated by shading). An uppercase letter within the consensus sequence indicates that at least 75% of the residues at that position are identical, while a lowercase letter indicates that at least 75% of the residues are conserved. Conserved amino acids are those that fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y); i (I, L, M, or V); and k (K or R). Highly conserved sequences conform to the consensus sequence at 75% or more of its positions. Variant sequences conform to the consensus sequence of the highly conserved group at 50 to 75% of the positions. Alternate blocks are derived from groups of proteins having a consensus sequence over that sequence block that differs from the corresponding highly conserved sequence at more than half of its positions. Novel sequences have no discernible homology to a conserved block that occupies the same relative position within sequences in the conserved group.

FIG. 2

Amino acid sequence blocks conserved among Cry proteins. For each block, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid (indicated by shading). An uppercase letter within the consensus sequence indicates that at least 75% of the residues at that position are identical, while a lowercase letter indicates that at least 75% of the residues are conserved. Conserved amino acids are those that fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y); i (I, L, M, or V); and k (K or R). Highly conserved sequences conform to the consensus sequence at 75% or more of its positions. Variant sequences conform to the consensus sequence of the highly conserved group at 50 to 75% of the positions. Alternate blocks are derived from groups of proteins having a consensus sequence over that sequence block that differs from the corresponding highly conserved sequence at more than half of its positions. Novel sequences have no discernible homology to a conserved block that occupies the same relative position within sequences in the conserved group.

FIG. 2

Amino acid sequence blocks conserved among Cry proteins. For each block, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid (indicated by shading). An uppercase letter within the consensus sequence indicates that at least 75% of the residues at that position are identical, while a lowercase letter indicates that at least 75% of the residues are conserved. Conserved amino acids are those that fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y); i (I, L, M, or V); and k (K or R). Highly conserved sequences conform to the consensus sequence at 75% or more of its positions. Variant sequences conform to the consensus sequence of the highly conserved group at 50 to 75% of the positions. Alternate blocks are derived from groups of proteins having a consensus sequence over that sequence block that differs from the corresponding highly conserved sequence at more than half of its positions. Novel sequences have no discernible homology to a conserved block that occupies the same relative position within sequences in the conserved group.

FIG. 2

Amino acid sequence blocks conserved among Cry proteins. For each block, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid (indicated by shading). An uppercase letter within the consensus sequence indicates that at least 75% of the residues at that position are identical, while a lowercase letter indicates that at least 75% of the residues are conserved. Conserved amino acids are those that fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y); i (I, L, M, or V); and k (K or R). Highly conserved sequences conform to the consensus sequence at 75% or more of its positions. Variant sequences conform to the consensus sequence of the highly conserved group at 50 to 75% of the positions. Alternate blocks are derived from groups of proteins having a consensus sequence over that sequence block that differs from the corresponding highly conserved sequence at more than half of its positions. Novel sequences have no discernible homology to a conserved block that occupies the same relative position within sequences in the conserved group.

FIG. 2

Amino acid sequence blocks conserved among Cry proteins. For each block, the consensus sequence denotes the positions at which at least 75% of the aligned proteins in the group have an identical or conserved amino acid (indicated by shading). An uppercase letter within the consensus sequence indicates that at least 75% of the residues at that position are identical, while a lowercase letter indicates that at least 75% of the residues are conserved. Conserved amino acids are those that fall into the following groups: a (A, G, S, T, or P); d (D, E, N, or Q); f (F, W, or Y); i (I, L, M, or V); and k (K or R). Highly conserved sequences conform to the consensus sequence at 75% or more of its positions. Variant sequences conform to the consensus sequence of the highly conserved group at 50 to 75% of the positions. Alternate blocks are derived from groups of proteins having a consensus sequence over that sequence block that differs from the corresponding highly conserved sequence at more than half of its positions. Novel sequences have no discernible homology to a conserved block that occupies the same relative position within sequences in the conserved group.

FIG. 3

Positions of conserved blocks among Cry proteins. The cartoon shows the sequence arrangement for each holotype toxin (e.g., Cry1Aa1) having at least one of the conserved blocks defined in the legend to Fig. 2. Sequence blocks are shown as dark gray, light gray, or white to indicate high, moderate, or low degrees of homology, respectively, to the consensus sequence for each conserved block. Variant (var) alternate (alt) are as defined in the legend to Fig. 2. The lengths of each protein and the conserved blocks within them are drawn to scale.

FIG. 4

Sequence similarity groups found among Cry and Cyt proteins. Sequences were aligned by using CLUSTAL W and a phylogenetic tree was constructed by NEIGHBOR as described in the accompanying work (79). The tree was visualized as a radial phylogram by using the TREEVIEW application. The proposed similarity groups are indicated by shading.

FIG. 5

Predicted three-dimensional structure of Cry1Ab highlighting the domain II residues shown by mutagenesis to be involved in receptor binding. Domains I (white), II (blue), and III (red) and portions of loops 1 (orange), 2 (yellow), and 3 (violet) and the α8 loop (green) are shown as space-filling molecular structures in the standard presentation (left) and rotated 90° (right).