Identification of a novel alternate promoter element in the pheST operon of Escherichia coli

Earlier work in this laboratory revealed that fitA was same as pheS as a recombinant clone, pSRJ5R1 harboring pheS⁺ gene complemented transcription-defective fitA76 Ts (temperature sensitive) mutant. However, this clone lacked the native promoter (NP) of pheST operon. A putative − 10 promoter like element was suggested to act as promoter in this clone. This work investigated the veracity of this putative promoter as well as its downstream regulatory region towards driving the pheS expression.

Plasmid clones with promoter-mutations or -deletions were constructed by PCR-based cloning and their ability to complement fitA76 Ts mutant strains was checked. Chromosomal mutations were transferred into various genetic backgrounds via P1-transductions. Relative viability assays were performed to check the extent of complementation.

Clones harboring point mutations (PM-pheS) or deletion (PD1-pheS) of − 10 region of the putative promoter did not abolish complementation of the fitA76 Ts phenotype. Subsequently, a novel alternate promoter (AP) was discovered by downstream deletion clone (PD2-pheS) which failed to complement. Keeping PD1-pheS intact but mutating initiation codon of pheS (ATG→TTG) failed to complement. Complementation ability of novel alternate promoter is poor in HfrC strain background unlike native promoter which complements well independent of strain background.

A novel alternate-promoter of pheST operon was identified by mutational/deletional analyses and earlier reported putative − 10 promoter was shown to be dispensable. Alternate promoter is relA dependent.