Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Abstract

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCE Bicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogen Pseudomonas aeruginosa This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

Keywords: Pseudomonas aeruginosa; Streptomyces; antibiotic; biosynthesis; gene transfer; phylogenetic analysis.

FIG 1

Bicyclomycin biosynthesis. (A) Simplified schematic of BCM biosynthesis. (B) bcm gene clusters identified in S. cinnamoneus and P. aeruginosa.

FIG 2

Heterologous expression of the bcm gene cluster from S. cinnamoneus in S. coelicolor M1146. Extracted ion chromatograms (EICs) of bicyclomycin (m/z 285.11, [M-H₂O+H]⁺) in S. cinnamoneus, S. coelicolor M1146 expressing the bcm cluster, and S. coelicolor M1146 containing empty vector. The intensity scale of each EIC is noted under the corresponding label.

FIG 3

Heterologous expression of the bcm cluster from P. aeruginosa in P. fluorescens SBW25. Base peak chromatograms of a bicyclomycin standard, P. fluorescens SBW25 expressing the bcm cluster, and P. fluorescens SBW25 containing empty vector. The intensity scale of the chromatograms is noted under the corresponding labels. Compounds produced by the heterologous expression strain but not found in the control strain are highlighted in gray.

FIG 4

Phylogeny and genetic context of the bcm gene clusters. (A) Unrooted maximum likelihood tree of the nucleotide sequences from the bcm gene clusters identified in this work. Branches are color-coded by genus, and major clades are highlighted. (B) Detailed view of the phylogeny and genetic context of the bcm clusters in Gram-positive bacteria. Bootstrap support values for the phylogeny are shown at the base of each branch, and the genetic context of each cluster (color-coded as in Fig. 1B) is shown for each branch of the tree. Flanking genes are color-coded gray if they encode proteins with conserved domains, white for hypothetical proteins with no conserved domains, and red for proteins related to mobile genetic elements (see Table S5 for details). Vertical black lines represent tRNA genes. (C) Genetic context of the bcm clusters in Gram-negative bacteria. The black triangle represents an attTn7 site.

FIG 5

Distribution of the bcm gene cluster across P. aeruginosa isolates using a modified version of the unrooted maximum likelihood tree generated by Kos et al. (38). The PA14-like, PA7-like, and PAO1-like clades are color-coded, and a green dot signifies the presence of the bcm gene cluster. P. aeruginosa SCV20265 and multiple reference strains (PAO1, M18, PA7, and PA14) are also labeled.

FIG 6

Maximum likelihood tree of the bcm 2-OG dioxygenases, including all representatives from Streptomyces, Actinokineospora (Actino), Williamsia (Will), Burkholderia (Burkh), and Tistrella (Tistr), two from Mycobacterium, and eight from Pseudomonas. Protein BP3529 from Bordetella pertussis was used as an outgroup (see Fig. S14 for an unrooted version of this tree). Background colors and numbering of the branch labels represent the position of a particular dioxygenase in the bcm gene cluster, as shown in the schematic representation. The taxonomic origins of each protein are indicated by their branch and label colors (black for Gram-positive and red for Gram-negative representatives). Bootstrap support values for the major branches are shown. The evolutionary relationships between bcm oxygenases from Gram-positive and Gram-negative bacteria are indicated by arrows in the diagram.