The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA

The entire gene cluster encoding enzymes involved in biosynthesis of L-tryp tophan in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 has been cloned and sequenced. Seven ORFs, which encode indole-3-glycerol phosp hate synthase (trpC), anthranilate phosphoribosyltransferase (trpD), the tw o subunits of anthranilate synthase (trpEG), phosphoribosyl :anthranilate i somerase (trpF) and the two subunits of tryptophan synthase (trpAB), were i dentified. The gene order is trpCDEGFBA, covering a region of 6045 bp. In o rder to confirm the function of the gene products, we expressed the first g ene, Pk-trpC, in Escherichia coli. The protein product was purified, and wa s found to show the expected indole-3-glycerol phosphate:synthase activity, with a temperature optimum of 85 degrees C. We could clearly identify a si ngle mRNA transcript by Northern analysis using probes in the central and 3 '-regions of the gene cluster, indicating that the gene cluster is transcri bed as an operon. A significant increase in try mRNA level was observed in cells grown in medium depleted of L-tryptophan, compared to cells grown in medium supplemented with L-tryptophan: indicating that expression of the ge ne cluster is regulated at the transcriptional level.