Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora

Abstract

Access to genome sequence data has challenged traditional natural product discovery paradigms by revealing that the products of most bacterial biosynthetic pathways have yet to be discovered. Despite the insight afforded by this technology, little is known about the diversity and distributions of natural product biosynthetic pathways among bacteria and how they evolve to generate structural diversity. Here we analyze genome sequence data derived from 75 strains of the marine actinomycete genus Salinispora for pathways associated with polyketide and nonribosomal peptide biosynthesis, the products of which account for some of today's most important medicines. The results reveal high levels of diversity, with a total of 124 pathways identified and 229 predicted with continued sequencing. Recent horizontal gene transfer accounts for the majority of pathways, which occur in only one or two strains. Acquired pathways are incorporated into genomic islands and are commonly exchanged within and between species. Acquisition and transfer events largely involve complete pathways, which subsequently evolve by gene gain, loss, and duplication followed by divergence. The exchange of similar pathway types at the precise chromosomal locations in different strains suggests that the mechanisms of integration include pathway-level homologous recombination. Despite extensive horizontal gene transfer there is clear evidence of species-level vertical inheritance, supporting the concept that secondary metabolites represent functional traits that help define Salinispora species. The plasticity of the Salinispora secondary metabolome provides an effective mechanism to maximize population-level secondary metabolite diversity while limiting the number of pathways maintained within any individual genome.

Keywords: comparative genomics; genome sequencing.

Fig. 1.

Enediyne pathway exchange. Three different OBUs were detected in the same chromosomal position in three different S. pacifica strains. These OBUs were classified as enediyne PKSs based on a NaPDoS analysis of the KS domains derived from the type I PKS genes (in red) in each pathway. The different OBU assignments are supported by the products of the sporolide (spo) and cyanosporaside (cya) gene clusters, which include sporolide A (1) and cyanosporaside A (2), respectively. These compounds, which are shown to the left of the pathways responsible for their production, possess fundamentally different carbon skeletons and are predicted to originate from enediyne precursors (28, 65, 66). Amino acid sequence identities relative to orthologs in the cya pathway are shown for representative genes. Products have yet to be identified from PKS32, which appears at the bottom.

Fig. 2.

Evolution of the cya pathway in S. pacifica. (A) The cya OBU contains three different versions of the pathway. The cya1 version is responsible for the biosynthesis of cyanosporaside A, which is derived from an enediyne precursor (28). It was observed in all strains in the uppermost clade of the Salinispora species tree (boxed in red). Two truncated versions of the pathway were observed, with cya2 appearing ancestral to cya1 in the species tree. Genes missing in cya2 and 3 include cyaA4 (O-acyltransferase), cyaN1 (epoxide hydrolase), and cyaN2 (oxidoreductase), which are predicted to encode tailoring enzymes. Together, the cya1- and 2-containing strains form a single clade (clade 1) in this region of the S. pacifica species tree. The cya3 pathway occurs in a separate S. pacifica lineage (clade 2). Products have yet to be identified from cya2 and 3. (B) A likelihood analysis predicts three independent acquisition events for the cya pathway, one in S. tropica and two in S. pacifica (red arrows). The S. pacifica acquisition events correspond to clades 1 and 2 in the species tree. (C) Maximum-likelihood phylogeny of the cyaE enediyne PKS gene including the top 10 BLASTp matches (bootstrap values for 100 replicates are shown at major nodes) reveals two major lineages (red arrows, numbers 1 and 2) that correspond to the strains in clades 1 and 2 of the species tree. This supports the vertical inheritance of this gene subsequent to acquisition. The position of the S. tropica (CNB-536) cyaE homolog within S. pacifica clade 1 suggests that the acquisition event in S. tropica is the result of horizontal gene transfer with S. pacifica clade 1. Gene gain and loss are assumed to account for the variations in the cya1–3 pathways.

Fig. 3.

Distribution and diversity of PKS and NRPS OBUs. (A) Rank-abundance curve showing the abundance of each OBU among the 75 strains analyzed (representative OBU names are shown). (B) Rarefaction curves with diversity estimators for each species.

Fig. 4.

OBU hierarchical cluster analysis and Salinispora species tree. Hierarchical cluster analysis based on OBU presence or absence. Maximum-likelihood species phylogeny generated from 10 housekeeping genes. Colors indicate the collection site: blue, Bahamas; pink, Palau; black, Fiji; red, Sea of Cortez; orange, Palmyra; turquoise, Guam; green, Hawaii.

Fig. 5.

Salinispora phylogeny depicting OBU inferred ancestry. A simplified species tree generated from 10 housekeeping genes (Fig. S4) shows 12 major Salinispora lineages with the number of strains in each indicated in blue adjacent to the node branch points. Boxes indicate the number of OBUs originating at various points in the species tree. Red, shared with a common ancestor of the genus; green, genus-specific; purple, shared with S. tropica and S. pacifica; black, species-specific; gray, clade-specific. Representative OBU names are indicated next to the point of acquisition. Orange arrows describe inter- and intraspecies OBU exchange events (cya exchange between S. tropica and S. pacifica is indicated in bold).

Fig. 6.

Linear pseudochromosomes reveal the positioning of Salinispora OBUs within genomic islands (numbered and shaded) as identified based on previously defined boundaries (24). Pathway positions were mapped using PKS and NRPS genes as reference. Only the pathways that could be mapped onto the pseudochromosomes are depicted. Pathways are color-coded and listed on the right next to the strain name and geographic origin. Singletons are depicted in light gray.