Solubilization, Activation, and Insecticidal Activity ofBacillus thuringiensisSerovar thompsoni HD542 Crystal Proteins

Abstract

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.Bacillus thuringiensis is a gram-positive bacterium, which during sporulation produces crystalline inclusions consisting of one or more insecticidal proteins known as delta-endotoxins. Based on sequence similarity, delta-endotoxins, mostly designated Cry proteins, may be divided into several broad classes (for a review, see reference 7). The vast majority of characterized Cry proteins show a number of conserved motifs, probably share similar structures, and therefore could tentatively be called three-domain Cry proteins. However, there exists a sofar small number of Cry proteins not related to the threedomain proteins, some of which can be arranged in small homology groups. These are the Cyt proteins, the Bin-like proteins, the Mtx2/3-like proteins, and the unique Cry6 and Cry22 proteins (9). These proteins may have very different modes of action compared to that of the 3-domain proteins and, therefore, are interesting subjects for further study.Cry15Aa from B. thuringiensis serovar thompsoni (5) is a member of the Mtx2/3-like group, due to its similarity to the mosquitocidal Mtx2 and Mtx3 proteins from Bacillus sphaericus. Other members of this group, which are more similar to Cry15 than are Mtx2 and Mtx3, are the Cry23, -33, -38, and -45 proteins from B. thuringiensis. Their amino acid sequences show weak similarity to ß-barrel pore...

Biochemical and molecular characterization of δ-endotoxins in Bacillus thuringiensis

“…Although proteinase cleavage occurs in the parasporal bodies, the lack of toxicity exhibited by the native strains (mainly in RT7 and RT24) could be explained as implying that the two insects are not the appropriate hosts (Du et al 1994); the insect midgut lacks suitable receptors for these kinds of toxins, low affinity of the crystals to vesicles in the membrane of apical microvilli, the binding of crystals in vivo is dependent on both the affinity and the concentration of receptor sites, or complete absence of specific binding sites for toxins (Van Rie et al 1990;Denolf et al 1993), which is thought to be the most important factor that affects toxic capability (Garczynski et al 1991). These results agree with that of Naimov et al (2008) in which a Cry15Aa crystal protein is ineffective against Helicoverpa armigera and Spodoptera exigua, while this same protein is highly toxic to Manduca sexta, Cydia pomonella and Pieris rapae.…”

“…Many studies have been conducted in order to correlate the biochemical and molecular properties of a crystal protein with its insecticidal activity (López-Meza and Ibarra 1996;Konecka et al 2007;Naimov et al 2008;Swiecicka et al 2008). Although a variety of crystals have been studied, there is not always a positive relation with the entomopathogenic activity (Benintende et al 1999).…”

Insecticidal activity of Bacillus thuringiensis crystal proteins

“…For the purpose of this review, a master list of all tested species was established, and output tables from plain language queries by species were manipulated in Microsoft Excel and aggregated at various levels. Data published since January 2008, providing toxicity information on Cry1Ab21 (Swiecicka et al, 2008), Cry1Ac17 (Hire et al, 2008), Cry1Ah (Xue et al, 2008), Cry1Ia (Martins et al, 2008), Cry2Ab10 (Lin et al, 2008), Cry2Af1 (Beard et al, 2008), Cry8Db (Yamaguchi et al, 2008), Cry5Ad (Lenane et al, 2008), Cry5Ba and Cry6Aa (Guo et al, 2008b), Cry9Bb (Silva-Werneck and Ellar, 2008), Cry15Aa (Naimov et al, 2008), Cry48Aa-49Aa (Jones et al, 2008), and Cry55Aa (Guo et al, 2008b), were included in the qualitative summaries only.…”

Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects

“…Besides that, Cry15Aa shares sequence similarity to mosquitocidal Mtx2 and Mtx3 protein, and shows hemolytic activity and toxicity to some types of Lepidopteran insects (Naimov et al, 2008). Strain 4AA1 genome codes some virulence factors, like two chitinases, metalloproteases InhA1 and InhA3, Bacillus enhancin-like protein Bel as well as non-hemolytic enterotoxin (NHE), enterotoxin FM (EntFM), and Hemolysin BL (HBL) (Raymond et al, 2010).…”