Molecular cloning and expression of the biodegradative threonine dehydratase gene (tdc) of Escherichia coli K-12

Summary. The biodegradative threonine dehydratase gene (tdc) of Escherichia coli was cloned by isolating a dehydra-tase-negative mutant after Tn5 mutagenesis, cloning the tdc::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoter-like elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P2, was located immediately upstream from the tdc coding region, and a second, P1, was approximately 1 kilobase upstream from P2. Deletion of the potential CAP-binding site from P1 prevented tdc gene expression. However, removal of P2 and a large seg-ment of the upstream DNA had no discernible ffect on dehydratase ynthesis. A 936-base pair open reading frame was found between Pl and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that Pa, and not Pz, is necessary for tdc gene expres-sion, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit