Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000

Abstract

Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.

FIG. 1.

Schematic of the targeted biosynthetic gene cluster in P. syringae pv. tomato DC3000. (Top) Graphic representation of the targeted gene cluster spanning pspto_2828 to pspto_2833. Colors of genes indicate putative functions of coded proteins: white, NRPS; gray, regulation; black, export. (Middle) Predicted NRPS domains, shown as circles, encoded by pspto_2829 and pspto_2830. Abbreviations: A, adenylation domain; C, condensation domain; Te, thioesterase domain; C*, dual-function condensation-epimerization domain. Domains have been grouped into modules, with each module identified by a bar and module number. (Bottom) Proposed lipopeptide sequence based on the order and predicted substrate specificity of A domains and the presence of an initiating C domain. The use of syf for the gene designation and Syf for the protein designation is proposed for those genes and proteins noted (see “Summary and conclusions,” below).

FIG. 2.

Phenotypes associated with the pspto_2828-pspto_2833 gene cluster. (A) Motility of P. syringae pv. tomato DC3000 strains on KB medium containing 0.5% agar. Five μl of cells from each strain was separately spotted to the center of a plate and subsequently incubated for 48 to 72 h at 28°C. (B) Droplet collapse results for 20-μl aliquots of cell-free supernatants of liquid cultures. Methylene blue was added to each sample to aid in visualization. (C) Silica gel TLC separation of ethyl acetate extractions of liquid cultures, visualized with bromothymol blue staining. The arrow identifies a region of the TLC plate where a metabolite from the wild-type (WT) strain reacted with the bromothymol blue stain, indicating a lipid-containing metabolite. Spc, spectinomycin resistance gene cassette.

FIG. 3.

Reverse-phase HPLC analysis of partially purified metabolites. Trace A, wild type; trace B, pspto_2828::spc; trace C, Δpspto_2829. Metabolite elution was monitored at 210 nm. The absorption peaks of the four samples analyzed further are highlighted with arrows, and the time of elution from the HPLC is noted.

FIG. 4.

Amino acid analysis. Data shown represent the numbers and identities of amino acids recovered after acid hydrolysis of metabolites collected at the elution times indicated. The data were normalized to a total of eight amino acids. GLX represents Gln or Glu.

FIG. 5.

Analysis of ESI-MS/MS fragmentation for the metabolite eluting at 24.1 min. (Top) A schematic is shown representing sites of cleavage in the proposed metabolite structure that would yield b and y ions. The theoretical masses of these ions are noted above the b notation and below the y notation. (Bottom) Ion masses obtained from ESI-MS/MS fragmentation of the [M + 2H]²⁺ form of the metabolite are shown. The observed ESI-MS/MS fragmentation ion masses are consistent with the theoretical values derived from the proposed lipopeptide structure as denoted at top. b° and y° denote dehydrated fragmentation products; b* denotes deaminated fragmentation product. The ion at 256.2 amu is consistent with the “a₂” ion fragment [3-hydroxydecanoyl + Leu − CHO].

FIG. 6.

Proposed structures of lipopeptide metabolites. (A) Summary of ESI-MS/MS data obtained from metabolites eluting from the HPLC column at the times indicated. Shown are the observed masses of b and y and dehydration (b°, y°) and deamination (b*) fragments. The observed masses are consistent with the theoretical values. ND, not detected. The R₁ and R₂ groups are explained in panel B. (B) Proposed structures of the six metabolites, syringafactin A to F, produced by P. syringae pv. tomato DC3000 based on amino acid analysis and mass spectrometry.