New Strategy For Isolating Novel Nematicidal Crystal Protein Genes From Bacillus Thuringiensis Strain YBT-1518

A novel strategy was developed for isolating cry genes from B. thuringiensis that uses an acrystalliferous B. thuringiensis host strain and a shuttle vector to construct a gene library, which is then screened by checking the formation of crystals by optical microscope observation and SDS-PAGE analysis. By this method, three cry genes cry55Aa1, cry6Aa2, and cry5Ba2 were cloned from long rice-shaped crystal producing B. thuringiensis YBT-1518, whose crystal protein shows toxicity to nematodes and consists of 54 and 45 kDa proteins as shown by SDS-PAGE. The 45 kDa protein-encoding gene cry55Aa1 has no significant similarity to previously published crystal protein genes. Proteins Cry55Aa1 and Cry6Aa2 separately formed long rice-shaped crystal in the acrystalliferous B. thuringiensis strain BMB171, while protein Cry5Ba2 was found to assemble as a bipyramidal crystal. Gene cry5Ba2 remained cryptic in strain YBT-1518 as shown by SDS-PAGE or microscope observation. Proteins encoded by these three genes are all toxic to the root-knot nematode Meloidogyne hapla. Both cry55Aa1 and cry6Aa2 were found to be located on a novel small plasmid, designated pBMB0228(17.7 kb).The complete 177,32-bp sequence of the small "Cry" plasmid pBMB0228 from nematicidal B. thuringiensis strain YBT-1518 was analyzed. In addition to the two known nematicidal Cry protein genes, a cry6A-like gene and also the similar gene of cry6Aa2's negative regulator gene R2, R2-like. The most interesting thing is that this plasmid encodes two kinds of Rep proteins and also two similar Mob proteins.By confirming the function of this plasmid, it was shown that both of the two Rep proteins could initiate the plasmid replication in the acrystalliferous B. thuringiensis host strain BMB171. The conjugal mating test revealed that recombinant plasmids harboring either of the two Mob protein genes could transfer from this host strain to another with the helper plasmid pAW63::Tn5401, a self-transmissible plasmid from B. thuringiensis kurstaki HD73 derivative AW48. And their transfer efficiency is similar to that of plasmid pBMBtl, pGI2 and also pTX14-3 from other B. thuringiensis strains.The investigation result showed that the high expression of gene cry55Aa1 and cry6Aa2 didn't affect the expression of cry5Ba2 in the wild-type B. thuringiensis strain YBT-1518. The nearest two orfs, upstream of cry5Ba2 gene and the nearest orf downstream the gene didn't contribute to its cryptic behavior either.