Identification of a polymyxin synthetase gene cluster of Paenibacillus polymyxa and heterologous expression of the gene in Bacillus subtilis

Abstract

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated pmxA, pmxB, pmxC, pmxD, and pmxE. The pmxC and pmxD genes are similar to genes that encode transport proteins, while pmxA, pmxB, and pmxE encode polymyxin synthetases. The insertional disruption of pmxE led to a loss of the ability to produce polymyxin. Introduction of the pmx gene cluster into the amyE locus of the Bacillus subtilis chromosome resulted in the production of polymyxin in the presence of extracellularly added L-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the pmx gene cluster is responsible for polymyxin biosynthesis.

FIG. 1.

The pmx gene cluster. (A) Genetic structure of pmx genes and domain organization of the Pmx enzymes. (B) Primary structure of polymyxin A. MOA, 6-methyloctanoic acid.

FIG. 2.

Analysis of polymyxin synthesis in P. polymyxa E681. LC analysis of culture supernatants of E681 (A) and the pmxE mutant (B), respectively, using a YMC Pack Pro C18 column. (C) MS data for polymyxin A produced by P. polymyxa E681. The arrow indicates the peak for polymyxin A. (D) Antibacterial activities of the culture supernatants of wild-type E681 and the pmxE mutant strains against E. coli DH5α.

FIG. 3.

Scheme for the transfer of pmx genes into B. subtilis. The pmx gene cluster was integrated into the amyE locus of B. subtilis BSK1 containing a deleted BsuM RM system from B. subtilis 168. The detailed protocol is described in Materials and Methods.

FIG. 4.

Antibacterial activities of recombinant B. subtilis strains against E. coli under conditions with or without l-Dab. B. subtilis BSK1 derived from B. subtilis 168 contains a deleted BsuM RM system. Strain BSK1S was constructed by introducing a functional sfp from B. subtilis CB114 into BSK1. BSK3 contains complete pmx genes (pmxABCDE) in the amyE locus of BSK1. BSK3S was constructed by introducing a functional sfp into BSK3.

FIG. 5.

Biosynthesis of polymyxin in B. subtilis. (A) LC analysis of culture supernatants of P. polymyxa E681 and B. subtilis BSK3S grown in GSC medium with or without l-Dab, using a Terra MS C18 column. Arrows indicate the peaks to be analyzed by MS. (B) MS data for polymyxins produced by P. polymyxa E681 and B. subtilis BSK3S.