Genetics of zwittermicin a production by Bacillus cereus

Abstract

Zwittermicin A represents a new chemical class of antibiotic and has diverse biological activities, including suppression of oomycete diseases of plants and potentiation of the insecticidal activity of Bacillus thuringiensis. To identify genes involved in zwittermicin A production, we generated 4,800 transposon mutants of B. cereus UW101C and screened them for zwittermicin A accumulation. Nine mutants did not produce detectable zwittermicin A, and one mutant produced eightfold more than the parent strain. The DNA flanking the transposon insertions in six of the nine nonproducing mutants contains significant sequence similarity to genes involved in peptide and polyketide antibiotic biosynthesis. The mutant that overproduced zwittermicin A contained a transposon insertion immediately upstream from a gene that encodes a deduced protein that is a member of the MarR family of transcriptional regulators. Three genes identified by the mutant analysis mapped to a region that was previously shown to carry the zwittermicin A self-resistance gene, zmaR, and a biosynthetic gene (E. A. Stohl, J. L. Milner, and J. Handelsman, Gene 237:403-411, 1999). Further sequencing of this region revealed genes proposed to encode zwittermicin A precursor biosynthetic enzymes, in particular, those involved in the formation of the aminomalonyl- and hydroxymalonyl-acyl carrier protein intermediates. Additionally, nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) homologs are present, suggesting that zwittermicin A is synthesized by a mixed NRPS/PKS pathway.

FIG. 1.

Chemical structure of zwittermicin A and the proposed precursors for its biosynthesis. The dashed lines delineate the individual precursors in the final molecule, while the arrows point to the corresponding precursor shown at the bottom. It is anticipated the 2,3-diaminopropionate component of zwittermicin A will be carbamoylated and amidated at some point after incorporation into the mixed nonribosomal peptide/polyketide backbone of the molecule.

FIG. 2.

Organization of genes identified in the zwittermicin A biosynthetic cluster of B. cereus UW85. Vertical arrows represent mutations within the designated gene. An open arrow indicates that the mutant produced zwittermicin A (52); a shaded arrow indicates that the mutant did not produce detectable levels of zwittermicin A (; this work). The domain organization of orf8 is indicated (see Results for details). Abbreviations: C, condensation; A, adenylation; PCP, peptidyl carrier protein; KS, ketoacyl synthase; AT, acyltransferase; KR, ketoreductase; ACP, acyl carrier protein.

FIG. 3.

Schematic representations of methoxymalonyl-ACP (a), aminomalonyl-ACP (b), and hydroxymalonyl-ACP (c) biosynthesis. The oval above each arrow indicates the enzyme proposed to catalyze the step being shown. Black and grey ovals are color coded to highlight homologous enzymes. The squiggled lines above FkbJ, ORF5, and ORF3 represent the 4′-phosphopentheinyl prosthetic group of each ACP. Question marks indicate enzymes or products yet to be identified.

FIG. 4.

Proposal for the minimum biosynthetic machinery needed for zwittermicin A assembly. The monomers incorporated into zwittermicin A are as follows: 1, serine; 2, malonyl; 3, aminomalonyl; 4, hydroxymalonyl; 5, 2,3-diaminopropionate. Each circle represents an individual domain of the NRPS/PKS megasynthase. The grey circles identify the ACP components of the aminomalonyl- or hydroxymalonyl-ACP precursors. The grey arrows indicate the direction of zwittermicin A assembly. The bars mark the borders between NRPS and PKS portions of the putative megasynthase. Abbreviations: A, adenylation; PCP, peptidyl carrier protein; KS, ketosynthase; KR, ketoreductase; AT, acyltransferase; C, condensation; AmT, amidotransferase; CT, carbamoyltransferase.