The expression and regulation of the Escherichia coli tdh operon and its role in the acquisition of the Tuc(+) phenotype

Benjamin David Aronson, Purdue University

Abstract

The TDH pathway of Escherichia coli converts threonine into glycine and acetyl CoA. This pathway can serve as an efficient alternative to the conventional (triose phosphate) pathway for serine and glycine biosynthesis. Selection of strains that are able to utilize threonine as a carbon and energy source (Tuc$\sp{+}$ phenotype) often gives rise to isolates with elevated levels of the TDH pathway enzymes. The genes that encode the two enzymes of the TDH pathway were sequenced. The two genes constitute an operon (designated the tdh operon) that has a transcription start site 26 nt upstream of the translational initiation site of the promoter proximal gene, kbl. The tdh promoter has a $-$35 hexameric sequence with weak homology to the consensus $-$35 hexamer sequence of $\sigma\sp{70}$ promoters. Mutations in the $-$35 region that either increased or decreased promoter strength were isolated and characterized. Transcription from the wild-type tdh promoter was induced about eight-fold upon growth in the presence of leucine. The cis-acting operator for leucine induction was localized in the interval between coordinates $-$69 and $-$35 relative to the transcription start site. In this region are two nucleotide segments that share some similarity with sequences in the upstream nontranslated regions of other leucine-regulated genes. Several constitutive tdh promoters generated by in vitro mutagenesis are described. The tdh promoter regions of eight Tuc$\sp{+}$ strains by selection in liquid culture were analyzed. Transcription from these promoters was constitutive and 100-fold elevated compared to the uninduced wild-type tdh promoter. It was determined that the transposable element IS3 had inserted, in an orientation and site-specific manner, into the tdh promoter regions of all eight Tuc$\sp{+}$ strains. The elevation of tdh expression in these Tuc$\sp{+}$ strains involved the formation of a hybrid promoter having a $-$35 hexamer situated within the IS3 element. The insertion event created a promoter of high signal strength while simultaneously disengaging cis-acting regulatory sequences originally present within the wild-type promoter. Using a procedure involving solid media for the selection of Tuc$\sp{+}$ mutants demonstrated that IS3 insertion was not obligatorily associated with the emergence of the Tuc$\sp{+}$ phenotype. Mutants having single nucleotide changes in the tdh promoter region were obtained as were Tuc$\sp{+}$ strains with unaltered tdh promoters. Some of the latter strains had mutations in the 80 minute region near mtl that was required for the expression of the Tuc$\sp{+}$ phenotype. A model for the mutational acquisition of the Tuc$\sp{+}$ phenotype is proposed that requires the mutational increases in activity of two steps involved in threonine metabolism.

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Biochemistry

COinS