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Using an oligonucleotide corresponding to the consensus sequence for the biotin-binding motif, two unlinked genetic loci, bpl1 and bpl2, were cloned from the erythromycin producer Saccharopolyspora erythraea and the nucleotide sequences of a c. 4 kb segment from each determined. The two loci share a virtually identical segment of 1746 nucleotides, coinciding with most of the genes designated bcpA1 and bcpA2 present in bpl1 and bpl2, respectively. The deduced sequences of these genes are highly similar to that of the α-chain of mammalian propionyl-CoA carboxylase. Upstream of bcpA2 lies pccB, the gene encoding the β-chain of this enzyme. Mutant strains carrying frameshift mutations in bcpA1 and pccB were constructed, but we failed to isolate insertional mutants in bcpA2. Propionyl-CoA carboxylase activity was undetectable in the pccB mutant, but was unaffected in the bcpA1-defective strain. These results indicate that pccB encodes the β-chain of propionyl-CoA carboxylases, and suggest that the α-chain of this enzyme, which is likely to be encoded by bcpA2, is shared with some other essential biotin-dependent enzyme. The pccB mutation had no impact on erythromycin production in complex medium.