THE TPL PROMOTER OF CITROBACTER-FREUNDII IS ACTIVATED BY THE TYRR PROTEIN

The ability of microorganisms to degrade L-tyrosine to phenol, pyruvat e, and ammonia is catalyzed by the inducible enzyme L-tyrosine phenol lyase (EC 4.1.99.2), To investigate possible mechanisms for how the sy nthesis of this enzyme is regulated, a variety of biochemical and gene tic procedures was used to analyze transcription from the tpl promoter of Citrobacter freundii ATCC 29063 (C, braakii), By computer analysis of the region upstream of the tpl structural gene, two segments of DN A bearing strong homology to the known operator targets of the TyrR pr otein of Escherichia coli were detected, A DNA fragment of 509 bp carr ying these operator targets plus the presumptive tpl promoter was synt hesized by PCR and used to construct a single-copy tpl-lacZ reporter s ystem, The formation of beta-galactosidase in strains carrying this re porter system, which was measured in E. coli strains of various genoty pes, was strongly dependent on the presence of a functional TyrR prote in, In strains bearing deletions of the tyrR gene, the formation of be ta-galactosidase was reduced by a factor of 10, Several mutationally a ltered forms of TyrR were deficient in their abilities to activate the tpl promoter, The pattern of loss of activation function was exactly parallel to the effects of the same tyrR mutations on the mtr promoter , which is known to be activated by the TyrR protein, When cells carry ing the tpl-lacZ reporter system were grown on glycerol, the levels of beta-galactosidase were 10- to 20-fold higher than those observed in glucose-grown cells, The effect was the same whether or not TyrR-media ted stimulation of the tpl promoter was in effect, By deleting the cya gene, it was shown that the glycerol effect was attributable to stimu lation of the tpl promoter by the cyclic AMP (cAMP) cAMP reporter prot ein system, A presumptive binding site for this transcription factor w as detected just upstream of the -35 recognition hexamer of the tpl pr omoter, The transcriptional start point of the tpl promoter was determ ined by chemical procedures, The precise locations of the TyrR binding sites, which were established by DNase I footprinting, agreed with th e computer-predicted positions of these regulatory sites, The two TyrR operators, which were centered at coordinates -272.5 and -158.5 with respect to the transcriptional start point, were independently disable d by site-directed mutagenesis, When the upstream operator was altered , activation was completely abolished. When the downstream operator,va s altered, there was a fourfold reduction in reporter enzyme levels, T he tpl system presents a number of intriguing features not previously encountered in TyrR-activated promoters, First among these is the ques tion of how the TyrR protein, bound to widely separated operators, act ivates the tpl promoter which is also widely separated from the operat ors.