Coronafacoyl Phytotoxin Biosynthesis and Evolution in the Common Scab Pathogen Streptomyces scabiei

Abstract

Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei Structural elucidation of accumulated biosynthetic intermediates in the ΔCYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria.IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.

Keywords: Pseudomonas syringae; Streptomyces scabiei; biosynthesis; coronafacoyl phytotoxin; coronatine; cytochrome P450; gene cluster; jasmonic acid; plant pathogens; signaling.

FIG 1

(A) Structure of the coronafacoyl phytotoxins coronatine (COR) and coronafacoyl-l-isoleucine (CFA-Ile) produced by P. syringae and S. scabiei, respectively, and of the bioactive plant hormone conjugate (+)-7-iso-jasmonyl-l-isoleucine (JA-Ile). (B) Organization of the CFA biosynthetic gene cluster from S. scabiei 87-22. Regulatory genes are in black, biosynthetic genes that have homologues in the P. syringae CFA biosynthetic gene cluster are in gray, and biosynthetic genes unique to the S. scabiei gene cluster are in white. The CYP107AK1 gene is highlighted in bold.

FIG 2

Proposed biosynthetic pathways for the production of coronafacoyl phytotoxins in P. syringae and S. scabiei. The hypothetical pathway in P. syringae is indicated by blue arrows, whereas the hypothetical pathway in S. scabiei is indicated by red arrows. Biosynthetic intermediates that have been isolated from S. scabiei biosynthetic mutants are in hatched boxes.

FIG 3

(A) RP-HPLC analysis of culture extracts from the S. scabiei ΔtxtA/pRLDB51-1 strain (blue line) and the ΔCYP107AK1 mutant (red line). The peak corresponding to the CFA-Ile coronafacoyl phytotoxin is indicated with an asterisk (*), and the peaks corresponding to minor coronafacoyl phytotoxins are indicated with a filled black circle (•). The two intermediates (i and ii) that accumulated in the ΔCYP107AK1 mutant extract are labeled. (B) Chemical structures of the purified intermediates i and ii. The carbon atoms are numbered according to the numbering scheme used for the raw data analysis (see Table S1 in the supplemental material). (C) Hypertrophy-inducing activity of organic culture extracts of S. scabiei ΔtxtA/pRLDB51-1 (IV) and ΔCYP107AK1 (V) on potato tuber tissue. Pure COR (1 nmol) was included as a positive control (II), while organic solvent (100% methanol; I) and culture extract from the CFA-Ile-deficient S. scabiei ΔtxtA Δcfa6 strain (III) were included as negative controls. The observed tissue hypertrophy is indicated by white arrows. (D) Hypertrophy-inducing activity of purified CFA-Ile (III) and of the ΔCYP107AK1 mutant intermediates i (IV) and ii (V) on potato tuber tissue. Solvent (methanol + 0.1% formic acid) served as a negative control (I), while pure COR (1 nmol) was used as a positive control (II). The observed tissue hypertrophy is indicated by white arrows.

FIG 4

(A) Heat map showing the protein BLAST identity hits to the S. scabiei 87-22 Cfa1-7 and Cfl proteins in different bacterial genomes. The actual percent amino acid identity is shown in each square. Abbreviations are as follows: Str, Streptomyces sp. NRRL WC-3618; Sgr, Streptomyces griseoruber DSM40281; Kaz, Kitasatospora azatica ATCC 9699; Azo, Azospirillum sp. B510, Psy, Pseudomonas syringae pv. tomato DC3000; Pam, Pseudomonas amygdali pv. morsprunorum, Pco, Pseudomonas coronafaciens pv. porri LMG 28495; Pps, Pseudomonas psychrotolerans NS274; Psa, Pseudomonas savastanoi pv. glycinea B076; Pat, Pectobacterium atrosepticum SCRI1043; Pbe, Pectobacterium betavasculorum NCPPB 2795; Pca, Pectobacterium carotovorum subsp. carotovorum UGC32; Bre, Brenneria sp. EniD312. (B) Organization of known and putative CFA biosynthetic gene clusters identified in sequenced bacterial genomes. Related genes are in the same color, and the known or predicted functions are indicated below. The genes that were used for construction of the concatenated phylogenetic tree (Fig. 6A) are denoted by an asterisk. Ssc, Streptomyces scabiei 87-22. (C) Domain organization of the Cfa6 and Cfa7 type I polyketide synthases encoded in the CFA biosynthetic gene clusters from different bacteria. The different modules (load, modules 1 and 2) are indicated as well as the enoyl reductase (ER) domain that is found only in a subset of Cfa7 proteins (indicated in orange). The domain nucleotide sequences that were used for construction of the concatenated phylogenetic tree (Fig. 6A) are denoted by an asterisk. Abbreviations are as follows: AT, acyltransferase; PP, phosphopantheinate; KS, ketosynthase; DH, dehydratase; KR, ketoreductase; TE, thioesterase.

FIG 5

NAD(P)H binding motifs from the Cfa7 ER domains of S. scabiei, Azospirillum sp. B510, K. azatica, S. griseoruber, and Streptomyces sp. WC-3618. The consensus NAD(P)H cofactor binding motif is in bold. Differences in motif sequences compared to the consensus sequence are in red.

FIG 6

Maximum likelihood phylogeny of the core CFA biosynthetic genes (A) and the 16S rRNA gene (B) from different bacteria. The CFA biosynthetic gene cluster tree was constructed using the concatenated cfa1–5, cfl, cfa6 (KS domain), and cfa7 (KS domain) nucleotide sequences. Bootstrap values of ≥50% are shown at the respective branch points and are based on 100 repetitions for the CFA gene cluster tree and 1,000 repetitions for the 16S rRNA gene tree. The scale bars indicate the number of nucleotide substitutions per site. Gammaproteobacteria are in red, Alphaproteobacteria are in blue, and Actinobacteria are in black. Known plant-pathogenic organisms are indicated with an asterisk (*). Abbreviations are as described for Fig. 4.