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:

The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism. Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level. In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

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The TrpI protein belongs to the LysR-family of procaryotic regulatory proteins. Members of this family share a characteristic similarity of their N-terminal amino acid sequences, and many of them are activators of divergently transcribed genes or operons. In Pseudomonas aeruginosa, the genes for tryptophan synthase, trpBA, are regulated by indoleglycerol phosphate (InGP) and TrpI. We demonstrate here that in the absence of InGP, the binding site of TrpI is located in the -52 to -77 region of the trpBA promoter; in the presence of InGP, the binding region is extended to the -32 region. In addition, two major, slow moving protein-DNA complexes are seen in gel retardation assays: the faster moving complex is formed in the absence of InGP and the amount of the slower moving complex is greatly enhanced in the presence of InGP. These results suggest that the binding of a second TrpI protein molecule, promoted by InGP, plays a crucial role in activating the expression of the trpBA gene pair.

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