CLONING AND CHARACTERISATION OF A GENE ENCODING A 100 KDA TOXIN FROM BACILLUS SPHAERICUS SSII-1 AND EXPRESSION OF INSECTICIDAL TOXINS IN CAULOBACTER CRESCENTUS

Abstract

A genomic library of B.sphaericus SSII-1 was constructed in the cosmid vector pHC79 and one recombinant cosmid, pKF2 was identified by screening the colonies for toxicity to Cu.quinquefasciatus larvae. A 3.8 kb Psti fragment derived from pKF2 inserted in pUCI 8 (pXP33 and pXP34, opposite orientations of the same insert) conferred toxicity to E.coli cells against Cu.quinquefasciatus and Ae .aegypti larvae. The nucleotide sequence of the 3.8 kb Pstl fragment translated into amino acid sequence showed the presence of one complete and one incomplete open reading frame. The complete open reading frame coding for a protein of 100 kDa has been identified as the toxin gene and it has been designated mtx (mosquitocidal toxin) gene. The putative promoters resemble vegetative promoters of B.subti/is consistent with the production of toxin before sporulation. Southern blot analysis shows that the mtx gene or a related gene is widely distributed in both high and low toxicity strains. The first 30 amino acids of the Mtx protein resemble a signal peptide and the amino acids 43 to 60 may represent a membrane spanning sequence. The N-terminal portion of the Mtx protein had strong regional homology with the region of pertussis toxin SI peptide identified to be essential for toxicity. The Mtx protein was also found to have three internal repeats in the C-terminal portion. The Mtx protein and its derivatives were purified using the glutathione Stransferase (GSD fusion vector system. Mtx protein minus the signal sequence (Mtx21, 97 kDa) was found to be toxic to Cu.quinquefasciatus larvae with an LC50 value of 15 ng/ml which is in the same order as that of the 51.4 kDa and 41.9 kDa binary toxins from B.sphaericus. Thus low toxicity of strain SSII-1 is probably due to low expression and/or low protein stability. The production of the 100 kDa toxin in E.coli is higher than in B.sphaericus SSII-1 cells, but SSII-1 cells were at least 10 times more toxic suggesting the presence of other unidentified toxins in B.sphaericus SSII-1. The purified 97 kDa protein was processed to a 27 kDa and 70 kDa protein by larval gut extracts and trypsin, while chymotrypsin completely degraded the protein. The N-terminal sequencing shows that both Cu.quinquefasciatus larval gut extract and trypsin have cleaved Mtx2 l (97 kDa) in the same region. The 70 kDa peptide is derived from the C-terminus while the 27 kDa peptide is de1ived from the N-terminus of Mtx21. The 2 proteins (27 and 70 kDa) could not be purified as active peptides by conventional methods. The proteins Mtx23 (74 kDa, a.a. 205 to 870), Mtx24 (32 kDa, a.a. 30 to 276), Mtx25 (57 kDa, a.a. 30 to 493) and Mtx26 (70 kDa, a.a. 259 to 870) were purified using the GST fusion system. The predicted molecular mass of Mtx24 peptide is 32 kDa but its mobility on SOS PAGE gave it an apparent molecular weight of 70 kDa. None of the peptides, Mtx23, Mtx24, Mtx25 and Mtx26 were toxic to Cu.quinquejasciatus larvae when fed individually. Mtx25 when fed together with Mtx23 or Mtx26 was found to be toxic while Mtx24 when fed together with either Mtx23 or Mtx26 was not toxic. This shows that both the 27 kDa peptide and the 70 kDa peptide are required for toxicity and part of the 70 kDa peptide sequence fused with the 27 kDa may be required for the proper folding of the 27 kDa peptide. Mtx21 (97 kDa) protein was found to ADP ribosylate itself and no additional bands were observed with total protein preparations from both HeLa and Cu.quinquefasciatus (07) cell line. Mtx24 (32 kDa) was unable to carry out any ADP ribosylation which could explain its lack of toxicity with Mtx23 (74 kDa) or Mtx26 (70 kDa). Mtx25 (57 kDa) ADP ribosylated itself as well as Mtx26 (70 kDa). Mtx25 also ADP ribosylated a 38 kDa protein and a 42 kDa protein in total protein, cytosol protein and membrane protein preparations from G7 cells. Mtx25 ADP ribosylated a 38 kDa protein in the total protein, cytosol protein and membrane protein preparations from HeLa cells. The correlation of ADP ribosylation with toxicity is not yet established. Mtx2 l (97 kDa) protein altered the morphology of Cu.quinquefasciatus (07) cells. Mtx21 had some effect on Ae.aegypti cells but had no effect on An.gambiae and HeLa cells. Mtx21 treated G7 cells lost their shape and fused with one another. Mtx26 (70 kDa) protein alone had the same effect as Mtx21 on 07 cells in contrast to the requirement of both the 70 kDa and the Mtx25 (57 kDa) peptide for toxicity to larvae. The 51.4 and 41.9 kDa toxin genes from B.sphaericus 2297 under the control of its native promoter (pJC3) and under the conu穙l of a regulatable tac promoter and native promoter (pJK69 and pJK73) in Cau/obacter crescentus CB15 were toxic to Cu.quinquefasciatus larvae. Ca.crescentus transformed with pJC3 were toxic to Cu.quinquefasciatus larvae giving 100% mortality with 3.7 X 107 cells/ml. Higher toxicity was obtained with Ca.crescentus cells transformed with pJK69 and pJK73. The 130 kDa toxin from B.thuringiensis subsp. israelensis under the control of tac promoter (pKH49) in Ca.crescentus had low toxicity (30% mortality after 48 hr) against Ae.aegypti larvae. The 100 kDa toxin from B.sphaericus SSII-1 under the native promoter (pSS6) in Ca.crescentus had low toxicity (30% mortality after 48 hr) to Cu.quinquefasciatus larvae.