Regulation ofcry gene transcription in Bacillus thuringiensis by a DNA binding protein which recognizes DNA elements upstream of the cryIAb, cryIC and cryID genes

Thomas M Walter, Purdue University

Abstract

The bacterium Bacillus thuringiensis produces during sporulation one or more proteinaceous crystalline inclusions with insecticidal properties. These protoxins are expressed from multiple cry genes located on large plasmids of 44 to 100 MDa. Each gene in a particular strain of B. thuringiensis may be expressed to a different extent, implying differential regulation. Two aspects of cry gene regulation were investigated. The role of each of the overlapping tandem promoters present in certain cry genes was examined. It appears that the upstream promoter, BtII, functions primarily to limit transcription from the downstream promoter, BtI. Mutations in conserved bases in the BtII promoter which maintained the spacing as well as the $-$10 and $-$35 sequences of the BtI promoter resulted in a four-to-five-fold increase in transcription of a lacZ fusion from the BtI promoter. A 350 bp region of DNA upstream of the cryIAb promoters was also examined. It was found that the presence of this region resulted in an additional four-fold increase in the rate and amount of transcription from the construct containing only the functional BtI promoter. This 350 bp region of DNA was retarded by cell extracts from several species and strains of sporulating Bacillus. A purified binding protein of apparent molecular weight 60 kDa was identified as the enzyme dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex. The protein bound specifically to the cryIAb upstream region as well as to the upstream regions from the related genes cryIC and cryID. The PDH-E2 gene from B. thuringiensis was cloned and purified as a glutathione-S-transferase fusion protein. This protein was shown to have binding activity and was recognized by B. subtilis anti-PDH-E2 antibody. The pure fusion protein protected three sites on the cryIAb DNA from digestion by DNAseI. Two of these sites share a consensus with other DNA sequences which are known or suspected binding sites for the PDH-E2 subunit. Therefore, it appears that the PDH-E2 protein may serve a dual role in B. thuringiensis: as a subunit of a multienzyme complex and as a sporulation-specific activator of certain cry gene transcription.

Degree

Ph.D.

Advisors

Aronson, Purdue University.

Subject Area

Molecular biology|Microbiology|Genetics

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS