We have found that Pseudomonas aeruginosa has two anthranilate synthase enzymes; they are homologous, and both have conventional ot and p subunits (9b). The two anthranilate synthases have different functions. One participates in tryptophan synthesis; its genes have been designated trpE and trpG (9b). These genes are quite similar to those of Pseudomonas putida (9a). In both pseudomonads, trpG, which encodes the p subunit, is cotranscribed with trpD and trpC in a three-gene operon.The second anthranilate synthase, previously misidentified as the product of a trp gene pair, was obtained on a R-prime plasmid by mating P. aeruginosa PAC174 carrying R68.44 with Escherichia coli W3110 tna AtrpE5 (8). This R-prime plasmid complements only those E. coli auxotrophs blocked in anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. Enzyme assays indicated that both the a and P subunits of anthranilate synthase were produced. The anthranilate synthase-encoding segment of the R-prime plasmid was subcloned into pBR322 (8). DNA sequencing of one of these subclones, pIA14, showed that the genes for the a and P anthranilate synthase subunits were indeed present on the cloned DNA. These genes are adjacent, and their coding sequences overlap by 23 base pairs * Corresponding author. t Present address:

In Pseudomonas aeruginosa, the trpI gene product regulates the expression of the trpBA gene pair encoding tryptophan synthase. trpI and trpBA are transcribed divergently. The trpI DNA sequence and deduced amino acid sequence were determined. The trpI start codon was found to be 103 base pairs from that of trpB. trpI encodes a 293-residue protein and the size of the trpI gene product, measured on sodium dodecyl sulfatepolyacrylamide gels, was close to that calculated from the amino acid sequence. The amino acid sequence of trpI resembles that of Enterobacter cloacae ampR, the regulatory gene for the ampC cephalosporinase. The N-terminal portions of trpI and ampR resemble corresponding portions of ilvY, metR, and lysR in Escherichia coli and nodD in Rhizobium meliloti. This resemblance may help to define a trpI-related family of activator proteins sharing a common structural plan.

We have determined the DNA sequence of the two adjacent genes for the alpha and beta chains of tryptophan synthase in Pseudomonas aeruginosa, along with 34 5'-flanking and 799 3'-flanking base pairs. The gene order is trpBA as predicted from earlier genetic studies, and the two cistrons overlap by 4 bp; a ribosome binding site for the second gene is evident in the coding sequence of the first gene. We have also determined the location of three large deletions eliminating portions of each gene. A detailed comparison of the deduced P. aeruginosa amino acid sequence with those published for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae shows much similarity throughout the beta and most of the alpha subunit. Most of the residues implicated by chemical modification or mutation as being critical for enzymatic activity are conserved, along with many others, suggesting that three-dimensional structure has remained largely constant during evolution. We also report the construction of a recombinant plasmid that overproduces a slightly modified alpha subunit from P. aeruginosa that can form a functionally effective multimer with normal E. coli beta 2 subunit in vivo.