The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization

The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requ irement for pyridoxal (pdx) and is a marker for the genetically mapped clus ter of genes associated with chloramphenicol biosynthesis. A gene regulatin g salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complement ation of the host's pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction an alysis indicated that the complementing plasmid had integrated into the chr omosome; after excision by a second crossover, the plasmid failed to propag ate. To avoid loss of the recombinant vector, a pdx-dependent Streptomyces lividans mutant, KAA11, with a phenotype matching that of S. venezuelae pdx -4, was isolated for use as the cloning host. Introduction of pIJ702 carryi ng an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant v ector containing a 2.5 kb S. venezuelae DNA fragment that complemented requ irements for pdx in both S. venezuelae and S. lividans mutants. Sequence an alysis of the cloned DNA located an intact ORF with a deduced amino acid se quence that, in its central and C-terminal regions resembled type-I aminotr ansferases. The N-terminall region of the cloned DNA fragment aligned close ly with distinctive helix-turn-helix motifs found near the N termini of Gnt R family transcriptional regulators. The overall deduced amino acid sequenc e of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genom e. This location is close to that of pdxA, the first pdx marker in S. coeli color A3(2) identified and mapped genetically in Sir David Hopwood's labora tory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be ho mologues regulating vitamin B6 coenzyme synthesis from pdx.