Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1 ( - ), cloN2 ( - ) and cloN7 ( - ) mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 ( - ) and cloN2 ( - ) mutants, but not in the cloN7 ( - ) mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.