1887

Abstract

The mutation in ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in was cloned by transforming a mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host’s pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdx-dependent mutant, KAA1, with a phenotype matching that of , was isolated for use as the cloning host. Introduction of pIJ702 carrying an genomic library into KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 25 kb DNA fragment that complemented requirements for pdx in both and mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix–turn–helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the A3(2) genome. This location is close to that of , the first marker in A3(2) identified and mapped genetically in Sir David Hopwood’s laboratory. The gene and are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.

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2001-08-01
2022-01-21
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