Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production

Abstract

As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the But R gene ( aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the But R gene. Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers). Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene ( erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production. Gene butB is transcribed in the same direction as but A and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide. Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated. It is proposed that ButB plays a role in the export of But from the producing organism.