REGULATION OF AN ANTHRANILATE SYNTHASE GENE IN STREPTOMYCES-VENEZUELAE BY A TRP ATTENUATOR

The nucleotide sequence of a 2.4 kb BamHI-SalI fragment of Streptomyce s venezuelae ISP5230 DNA that complements trpE and trpG mutations in E scherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two s ubunits of anthranilate synthase. The two-thirds nearest the amino ter minus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small O RF encoding 18 amino acids that include three adjacent Trp residues; i n addition the ORF contains inverted repeats with sequence and positio nal similarity to the products of attenuator (trpL) regions that regul ate tryptophan biosynthesis in other bacteria. In cultures of a trpC m utant of S. venezuelae, increasing the concentration of exogenous tryp tophan decreased the formation of anthranilate synthase; similar evide nce of endproduct repression was obtained in a trpCER mutant of E. coi l transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrat ions of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutati on in E. coli. However, S. venezuelae transformants in which the two-b ase deletion had been introduced by replacement of homologous chromoso mal DNA did not exhibit a Trp(-) phenotype, The result implies that S. venezuelae has one or more additional genes for anthranilate synthase . In alignments with anthranilate synthase genes from other organisms. ORF2 from S. venezuelae most closely resembled genes for phenazine bi osynthesis in Pseudomonas. The results bear on the function of the gen e in S. venezuelae.