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research-article

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli.

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.

ABSTRACT

The tdh operon of Escherichia coli consists of two genes whose products catalyze sequential steps in the formation of glycine and acetyl coenzyme A from threonine. The operation of the tdh pathway can potentially confer at least two capabilities on the cell: the first is to provide a biosynthetic source of glycine, serine, or both that is an alternative to the conventional (triose phosphate) pathway; the second is to enable cells to utilize threonine as the sole carbon source. The latter capability is referred to as the Tuc+ phenotype. In wild-type E. coli, the tdh operon is expressed at levels that are too low to bestow the Tuc+ phenotype, even in leucine-supplemented media, where the operon is induced eightfold. In eight Tuc+ mutants, the expression of the tdh operon was elevated 100-fold relative to the uninduced wild-type operon. The physical state of the DNA at the tdh locus in these Tuc+ strains was analyzed by Southern blotting and by DNA sequencing. In eight independent isolates the mobile genetic element IS3 was found to lie within the tdh promoter region in identical orientations. In six cases that were examined by DNA sequencing, IS3 occupied identical sites between the -10 and -35 elements of the tdh promoter. The transcription start points for the wild-type tdh promoter and one IS3-activated tdh promoter were identical. In effect, the repeatedly observed transposition event juxtaposed an IS3-borne -35 region and the tdh-specific -10 region, generating a hybrid promoter whose utilization led to elevated, constitutive expression of the tdh operon. This is the first case of promoter activation by IS3 where the site of transcription initiation is unaltered.