Effect of accessory proteins P19 and P20 on cytolytic activity of CytlAa from Bacillus thuringiensis subsp. israelensis in Escherichia coli (original) (raw)

2001, Current Microbiology

The gene coding for the accessory protein P19 of Bacillus thuringiensis subsp. israelensis was expressed in Escherichia coli and its product was characterized. To investigate its putative role in ␦-endotoxin crystallization as a P20-like polypeptide, each of the two encoding genes, p20 and p19, was cloned for inducible expression coordinatively with cyt1Aa. The latter is known to kill its transgenic host. P20 but not P19 stabilized Cyt1Aa and protected the host cells from its lethal effect. Neither GroEL nor GroES, expressed in trans, affected Cyt1Aa as did P20. The function of P20 is thus more specific than that of the chaperones, but that of P19 remains enigmatic. The correct sequence of p19, confirmed in all five isolates of B. thuringiensis subsp. israelensis, does not explain the slow electrophoretic mobility of its 179 amino acids product. Bacillus thuringiensis is a gram-positive soil bacterium that produces during sporulation [27, 36] large amounts of proteins in the form of crystals (␦-endotoxin). The insecticidal crystal proteins (ICPs) are toxic against a wide variety of insect larvae, mostly of Lepidopteran, Coleopteran, and Dipteran species. The ability to accumulate such quantities of these proteins within the cell is mainly due to their precipitation and aggregation, thus rendering them protease-resistant [5]. All subspecies include at least one Cry polypeptide (for crystal) of 65-145 kDa, and some (with anti-Dipteran specific activity) contain in addition a smaller protein, Cyt (for cytotoxic), of 25-28 kDa. The latter are less specific, hemolytic, and cytotoxic in vitro [22, 39], and not homologous to any of the Cry toxins, but may share a similar mechanism involving colloid-osmotic lysis [24, 25]. The C-terminal half of the large ICPs (125-145 kDa) is conserved and participates in crystal formation. These polypeptides spontaneously form biologically active inclusions probably via inter-and intra-molecular disulfide bonds [2, 3, 9, 14, 36]. The smaller ICPs (Ͻ 70 kDa), on the other hand, do not possess this conserved domain [22] and may thus require assistance in crystal formation. There is evidence that Cry2Aa (71 kDa) from B. thuringiensis subsp. kurstaki, and Cry11Aa (72 kDa) and Cyt1Aa (27 kDa) from B. thuringiensis subsp. israelensis, require the accessory proteins Orf2 and P20, respectively, for crystallization [1, 15, 32, 40, 46]. The apparent similarity in interactions between the Cry2Aa/ Orf2 and Cry11Aa/P20 systems is reflected in their gene organization. They are organized in an operon and cotranscribed with another gene not involved in toxicity, orf1 and p19, respectively [5, 17, 43]. The function(s) of the latter is(are) not known. The presence of P20 seems to raise the levels of Cyt1Aa and Cry11Aa by post-translational stabilization in recombinant Escherichia coli [1, 40] and acrystalliferous strains of B. thuringiensis [12, 13, 45, 46]. Similarly, disruption of orf2 leads to dramatic reduction of Cry2Aa and lack of Cry2Aa crystals in its original host B. thuringiensis subsp. kurstaki [15]. Moreover, expression of cloned cyt1Aa is lethal to E. coli [19] and B. thuringiensis subsp. kurstaki (cryB) cells [45]. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of recombinant E. coli cells