p-aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase

Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. v enezuelae. The second closely resembled the conserved pabB sequence consens us and hybridized with a 3.8 kb Ncol fragment of S. venezuelae ISP5230 geno mic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected thre e ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bact eria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 produc t resembled PabA of other bacteria. ORF3 was incomplete; its deduced partia l amino acid sequence placed it in the MocR group of GntR-type transcriptio nal regulators. Introducing vectors containing the 3.8 kb Ncol fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into t he Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosyn thesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These re present a second set of genes encoding 4-amino-4-deoxychorismate synthase i n S. venezuelae ISP5230. In contrast to the fused pabAB set previously isol ated from this species, they do not participate in chloramphenicol biosynth esis, but like pabAB they can be disrupted without affecting growth on mini mal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.