A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis.
Mutagenesis of Streptomyces venezuelae ISP5230 and selection for P-aminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants (VS519 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQ102 or pDQ103, which carried a pab-complementing fragment from S. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519. The cloned S. venezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHl-Sacl fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N- and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. venezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 micrograms mL-1 and blocked Cm production.
Brown
Michael P
MP
Department of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4J1Canada.
Aidoo
Kwamena A
KA
Department of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4J1Canada.
Vining
Leo C
LC
Department of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4J1Canada.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002174:Candicidin / Q000096:biosynthesis; D002701:Chloramphenicol / Q000096:biosynthesis*; D002827:Chorismic Acid / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D000637:Transaminases / Q000235:genetics / Q000502:physiology*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002174:Candicidin; D002701:Chloramphenicol; C022309:PABA synthase; D000637:Transaminases; D002827:Chorismic Acid