Gene transfer system in bacillus thuringiensis subsp. Israelensis by conjugation-like process

Abstract

Protoplast of a lysozyme sensitive strain of Bacillus sp. strain o 016 was transformed by plasmid pTF6 and pHV33 with the frequencies of 3.2 x 103 and 2.0 x103 transformants/ug DNA respectively. The plasmids pTF6 and pHV33 in Bacillus sp. strain 0 016 was subsequently transfered to Bacillus thuringiensis subsp. israelensis strain A084 (B.t.i.A084) by conjugation-like process with the frequencies of 3.5 x 10-7 and 2.5 x 10-7 (number of transcipients per number of recipients) respectively. Plasmid pTF6 was found to be stably maintained in Bacillus SP. strain 0 016 and B.t.i.A084 but the plasmid pHV33 was found to be unstable in Bacillus sp. strain.o 016 and B.t.i.A084. Recombinant plasmids pTF6-Xd and pHV33-Xd which were constructed by ligation between plasmid pTF6 or pHV33 and the xylosidase gene were transfered into Bacillus sp. strain o 016. It was found that plasmids pTF6-Xd and pHV33-Xd were not stable in either hosts. Another recombinant plasmids pBA401 which composed of plasmid pTF6 and the pencillin acylase gene was transferred into Bacillus sp. strain o 016 and subsequently into B.t.i.A084 with the frequencies of 9.9 x 102 transformants/ug DNA and 6.7 x 10-7 (number of transcipients per number of recipients) respectively. Plasmid pBA401 was found to be stable n both Bacillus sp. strain o 016 and B.t.i.A084. Studies by using bybridization technique indicate the presence of plasmid pBA401 in Bacillus sp. strain o 016. The gone for penicillin G acylase was also found to express in both Bacillus sp. strain o 016 and B.t.i.A084 hosts.