DIRECTED MUTAGENESIS OF A REGULATORY PALINDROMIC SEQUENCE UPSTREAM FROM THE BREVIBACTERIUM-LACTOFERMENTUM TRYPTOPHAN OPERON

A cloned 9.6-kb fragment of Brevibacterium lactofermentum DNA, carryin g the entire trp operon and upstream regulatory sequences, produces a polycistronic 7.0-kb transcript as detected by hybridization with an i nternal probe. The transcription start point (tsp) was identified by S 1 mapping. The operator-promoter (OF) region subcloned in Escherichia coli and B. lactofermentum promoter-probe vectors exhibited about tenf old higher activity in B. lactofermentum. A 14-bp wild-type (wt) palin drome located at bp -15 to -28 was mutated to change the conserved ade nine adjacent to the axis of symmetry. The wt and mutated OP regions w ere coupled to the amy reporter gene (encoding alpha-amylase [[Amy]) o r to the 5' region (trpE and trpG genes) of the trp operon, for expres sion 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 gro wn in minimal medium. Tryptophan repressed the OP as assayed by the an thranilate synthase (AS) activity in B. lactofermentum in construction s with the wt OP region, but surprisingly, caused a large stimulation of either AS or the Amy reporter activity, in constructions with the m utated OF. The palindromic sequence is, therefore, involved in a dual repression-stimulation control of expression of the trp operon.