Directed mutagenesis of a regulatory palindromic sequence upstream from the Brevibacterium lactofermentum tryptophan...

A cloned 9.6-kb fragment of Brevibacterium lactofermentum DNA, carrying the entire trp operon and upstream regulatory sequences, produces a polycistronic 7.0-kb transcript as detected by hybridization with an internal probe. The transcription start point (tsp) was identified by S1 mapping. The operator-promoter (OP) region subcloned in Escherichia coli and B. lactofermentum promoter-probe vectors exhibited about tenfold higher activity in B. lactofermentum. A 14-bp wild-type (wt) palindrome located at bp -15 to -28 was mutated to change the conserved adenine adjacent to the axis of symmetry. The wt and mutated OP regions were coupled to the amy reporter gene (encoding alpha-amylase [Amy]) or to the 5' region (trpE and trpG genes) of the trp operon, for expression studies. Constructions with the regulatory signals coupled to the wt trpE-trpG genes were introduced in a B. lactofermentum trpE mutant (obtained by gene disruption). The mutation in the palindrome did not affect the promoter activity in B. lactofermentum or E. coli when grown in minimal medium. Tryptophan repressed the OP as assayed by the anthranilate synthase (AS) activity in B. lactofermentum in constructions with the wt OP region, but surprisingly, caused a large stimulation of either AS or the Amy reporter activity, in constructions with the mutated OP. The palindromic sequence is, therefore, involved in a dual repression-stimulation control of expression of the trp operon.