Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2003
Publication Date: 3/1/2004
Citation: Moyne, A., Cleveland, T.E., Tuzun, S. 2004. Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. Federation of European Microbiological Societies Microbiol Letters. 234:43-49. Interpretive Summary: Methods need to be developed through research to prevent aflatoxin contamination of crops before harvest, since the presence of this fungal toxin in even very low amounts makes foods and feeds unacceptable for animal and human consumption due to its toxic and carcinogenic properties. One strategy to prevent aflatoxin development in plants is through the insertion of genes into plants which encode peptides (small proteins) inhibitory to the fungus that makes aflatoxin. As a basis for cloning these antifungal (inhibitory to fungal growth) genes, the inhibitory lipopeptide (a small protein with a lipid attached) called bacillomycin D produced by the bacterium, Bacillus subtilis, has been characterized. This accomplishment provided the basis for the present study, the cloning and the complete characterization of a cluster of genes (named BamA, BamB, BamC, and BamD) governing synthesis of bacillomycin D. Two genes that might be involved in regulation of bacillomycin D production were found to be located in the flanking region of the bacillomycin D gene cluster. Major scientific impact in the control of several plant diseases could result if potent antifungal genes can be utilized in biotechnology to limit infection of crops by fungal pathogens.
Technical Abstract: Bacillus subtilis AU195 produces bacillomycin D, a cyclic lipopeptide that is an inhibitor of the aflatoxin producing fungus, Aspergillus flavus. This inhibitor belongs to the iturin family that shares the common amino acid sequence Asx-Tyr-Asx linked to a B-amino fatty acid. Based on the DNA sequence similarities between bacillomycin D and mycosubtilin, we isolated and characterized the bacillomycin D operon consisting of 4 ORFs. Disruption of ORF 2, which links the amino acid moiety to the B-amino fatty acid, resulted in the loss of antifungal activity. The bacillomycin D operon is comprised of BamD, BamA, BamB, and BamC spanning 37.5 Kb. It shares the structural organization of the peptide synthetases. The DNA sequences of BamD, BamA, and the two modules encoding the activation of Tyr and Asn in BamB, are 98% identical to the corresponding DNA sequences of ItuD, ItuA, and ItuB. In contrast, the bacillomycin D ORF, BamC, shared more homology with the mycosubtilin operon than the iturin A operon. The proline module of BamB had closer homology to the proline module of the plipastatin synthetase of B. subtilis 168 (57%) than to the proline module of ItuB and MycB (47%). These results suggest that intergenic module replacement might have occurred between B. subtilis lipopeptide synthetase, including the iturin family and the plipastatin and fengycin family. In the flanking region of the bacillomycin D operon, we identified a two-component regulatory system that might be involved in the regulation of the operon.