Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1

Abstract

The 97-kDa protein Mtx2l, derived from the 100-kDa mosquitocidal protein (Mtx) from BaciUus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography. The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein. The 97-kDa Mtx21 protein was found to be toxic to Cukx quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml. Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa. Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern. N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein. The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.Bacillus sphaericus is an aerobic, gram-positive, sporeforming bacterium which is widespread in soil and aquatic environments. Some strains of B. sphaericus have been found to be pathogenic to mosquito larvae (14), and the pathogenicity of these bacteria has been attributed to the expression of toxic proteins (26,29,31,32 The high-toxicity strains have been shown to express a pair of proteins with relative mobilities corresponding to molecular masses of 63 and 43 kDa (5). Later studies in which the genes encoding these proteins were cloned and sequenced showed the molecular masses to be 51.4 and 41.9 kDa (2, 19). Both these proteins are required for toxicity to mosquito larvae (7, 13) and are expressed predominantly during sporulation (27). B. sphaericus SSII-1 is a lowtoxicity strain isolated from mosquito larva cadavers collected in India (29). The genes encoding the 51.4-and 41.9-kDa binary toxin have been shown to be absent from B.sphaericus . In order to study the nature of toxicity of B. sphaericus SSII-1, we have cloned a gene encoding a 100-kDa protein and have designated it the mix (mosquitocidal toxin) gene (31). The N-terminal region of the Mtx protein was found to have significant homology with the catalytic subunits of ADP-ribosyltransferase toxins, such as the pertussis toxin S1 subunit (28), and had no significant homology with the pair of toxins from the high-toxicity B. sphaericus strains (31). The aim of the present study was to purify the Mtx protein and to understand the processing of this protein in mosquito larvae to gain further insight into its mode of action. MATERIALS AND METHODSMaterials and strains. pGEX-1 and pUC18 were obtained from Pharmacia. Glutathione agarose, thrombin, and trypsin were purchased from Sigma USA. Escherichia coli (DH5) * Corresponding author. cells harboring plasmids were grown in Luria broth (24) supplem...

Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides

“…In vitro ADP-ribosylation was carried out with Mtx21 (10 pug), Mtx25 (5.7 pug), and Mtx24 (Fig. 3a, lane 2), corresponding to the Mtx2l and its breakdown product (31). There were no additional bands when Mtx2l was incubated with G7 total proteins (Fig.…”

“…The Mtx25 and Mtx26 proteins were found to migrate according to their predicted molecular masses, whereas Mtx24 was found to have a mobility corresponding to a 70-kDa protein. The purified proteins, Mtx26 (0.7 pug), Mtx24 (0.3 [ug), and Mtx25 (0.6 pug), were tested for toxicity against C. quinquefasciatus larvae as described elsewhere (31). None of the peptides were toxic to larvae on their own.…”

“…Larvicidal assays were performed as described previously (31). When combinations were used, larvae were exposed to test peptides in equimolar concentrations.…”

“…la). The ClaI*-AflIII * junction was a 30 100 200 300 400 500 600 700 8000870 I~4 GST 30 u8aP -8'0 I 4~~I GJST 30 493 b p13124 C ATOG CGA TIT ACT AGC CIT CTT MAA TIT TA MAA AAG ATOG MT TAT CAA TIT MAT TT0 CCG TM MAA TIT AlT TAT ACA ATr CAT COT GAC TGA AFIIII (31), and purification of Mtx protein derivatives was carried out as described by Smith and Johnson (29).…”

“…The plasmid pTH21 (Fig. la) (31) was digested with AflIII (which has three sites in the plasmid), and the cohesive ends were made blunt with DNA polymerase I Klenow fragment. The products were digested with NcoI, and the 0.7-kb NcoIAflIII* (* denotes a blunt-ended site) fragment was isolated, purified, and ligated into pTH21 prepared as follows.…”