Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa

Abstract

In Pseudomonas aeruginosa, the operon encoding tryptophan synthase (trpBA) is positively regulated by the TrpI protein and an intermediate in tryptophan biosynthesis, indoleglycerol phosphate (InGP). A gene fusion in which the trpBA promoter directs expression of the Pseudomonas putida xylE gene was constructed. By using a P. putida F1 todE mutant carrying this fusion on a plasmid, three cis-acting mutations that increased xylE expression enough to allow the todE strain to grow on toluene were isolated. The level of xylE transcript from the trpBA promoter was increased in all three mutants. All three mutations are base substitutions located in the -10 region of the trpBA promoter; two of these mutations make the promoter sequence more like the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. The activities of the wild-type and mutant trpBA promoters, as monitored by xylE expression, were assayed in P. putida PpG1 and in E. coli. The up-regulatory phenotypes of the mutants were maintained in the heterologous backgrounds, as was trpI and InGP dependence. These results indicate that the P. aeruginosa trpBA promoter has the key characteristics of a typical E. coli positively regulated promoter. The results also show that the P. aeruginosa and P. putida trpI activator gene products are functionally interchangeable.