Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

Abstract

Background: Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including alpha-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.

Results: Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to NPS genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.

Conclusions: This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the alpha-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.

Figure 1

Cartoons of tree topologies showing major NRPS subfamilies. All trees reflect phylogenetic analyses of the complete A domain dataset. A. NJ tree using a ML distance matrix created using the WAG plus gamma model. B. ML tree (PhyML) using the WAG plus gamma model. C. ML tree (RAxML) using the RTREVF plus gamma model. Bootstrap support greater than 50% is shown under branches. The light blue rectangle indicates primarily mono/bi-modular NRPS; the SID and EAS subclasses are primarily multimodular. Color coding for subfamilies: brown: adenylating enzyme outgroups; light green: fungal PKS;NRPS hybrid synthetases (PKS;NRPS); dark orange: ChNPS11/ETP module 1 synthetases (ChNPS11/ETP mod 1); dark blue: ChNPS12/ETP module 2 synthetases (ChNPS12/ETP mod 2); yellow: ChNPS10-like synthetases (ChNPS10); light blue: Cyclosporin synthetases (CYCLO); pink: α-aminoadipate reductases (AAR); dark green: ACV synthetases (ACV); red: siderophore synthetases (SID); purple: Euascomycete clade synthetases (EAS). The majority of bacterial sequences (dark gray) group together although they contain a few fungal A domains (ACV synthetases and the NRPS;PKS hybrid (ChNPS7;PKS24) suspected of being horizontally transmitted from bacteria to fungi. The remaining bacterial A domains group with the mono/bi-modular AAR and ChNPS12/ETP mod 2 subfamilies.

Figure 2

ML phylogenetic tree (PhyML, WAG plus gamma) from the reduced A domain dataset. Branches corresponding to subfamilies are color coded as in Fig. 1 and known products of NRPSs within each subfamily are shown to the right in parentheses. All C. heterostrophus NRPS A domains are indicated as red dots. Bootstrap values greater than 50% are shown above branches, where legibility makes this possible. This analysis shows stronger bootstrap support (97%) for grouping the exclusively fungal, multimodular subfamilies, SID and EAS subfamilies together (arrow). Double arrow indicates high bootstrap support (>85%) for grouping ChNPS11/ETP/ChNPS12 together.

Figure 3

Lineage specific expansions and contractions in number of NRPS genes per genome. Inferred number of NRPS encoding genes at ancestral nodes mapped onto the ultrametric tree of fungi. Timescale in millions of years is shown along bottom. Branches with significant expansions (blue) or contractions (red) are shown with associated p-values above branches. The largest contraction in number of NRPSs occurs in N. crassa while the largest expansion occurs in the ancestor of euascomycetes. A highly significant expansion also occurs in F. graminearum and significant expansions occur in several other euascomycete taxa (e.g., M. oryzae and on the branch leading to the Aspergillus species).

Figure 4

Hypothesized origins of major fungal NRPS subfamilies based on the oldest member of each subfamily. Subfamilies color coded as in Fig. 1. AAR, and ChNPS11/ETPmodule 1 and ChNPS12/ETP module 2 likely originated prior to or early in the divergence of fungi. AAR genes are present in all fungi, while the ChNPS11/ETP/ChNPS12 clade contains representatives of the most ancestral fungal group, the Chytridiomycota. as well as bacterial sequences that nest with high bootstrap support within this clade. Although ACV genes are clearly present in eubacteria, they appear to have been horizontally transferred to euascomycete fungi, hence their dual placement. The CYCLO, ChNPS10, and SID subfamilies were found in Basidiomycota, Schizosaccharomycota, and Euascomycota and thus likely originated in an ancestor of the Dikarya. Fungal PKS;NRPS hybrids and EAS were found only in Euascomycetes.

Figure 5

Conserved domain architectures for mono-bimodular NRPS subfamilies. The majority of mono-bimodular subfamilies have an A-T domain structure followed by various C-terminal domains. Only ChNPS11/ETP module 1 and ETP module 2 show complete A-T-C modules. The ChNPS12/ETP module 2 subfamily also contains representatives consisting of a single A domain. Domains: A = adenylation, T = thiolation, C = condensation, R = thioester reductase, D = ADH short chain dehydrogenase, PKS = polyketide synthase module, FeR = ferric reductase, FSH/SH = serine hydrolase, RH = polynucleotidyl transferase, Ribonuclease H, LPS = LPS-induced tumor necrosis alpha factor.

Figure 6

Phylogeny of the ChNPS11/ETP/ChNPS12 subclade. Extracted from maximum likelihood (PhyML with WAG plus gamma substitution matrix) phylogeny of complete A domain dataset (Additional file 6B). Domain structure of each NRPS is shown to the right of species abbreviation and accession number. Orange highlighted A domains reflect corresponding A domain in the phylogeny. Orange branches = ChNPS11/ETP mod1 and blue = ChNPS12/ETP mod2 subfamilies. ChNPS11 is monomodular, while all other NRPSs in the ETP module 1 group are bimodular; all have complete A-T-C modules. The A domain from a M. oryzae NRPS;PKS (MG07803.6) also groups here. Members of the ChNPS12 subfamily show a diversity of C-terminal domains as described in text and Fig. 5. The group includes two putative NRPSs from the chytrid, B. dendrobatidis, two proteins with either an incomplete (MGG15248.6) or a degenerate (BC1G07441_07442.1) first module and monomodular bacterial proteins consisting of single A domains. ChNPS12 homologs in Basidiomycete NPS12 group 2 consist of proteins with single A domains which appear to lack additional C-terminal domains and are highly expanded in the basidiomycete Postia placenta.

Figure 7

Phylogenetic analysis of the Euascomycete subclade. Tree extracted from the maximum likelihood (PhyML with WAG plus gamma substitution matrix) phylogeny of the complete A domain dataset (Additional file 6B). Branches defining subgroups of the EAS clade grouping with a C. heterostrophus NRPS A domain or with A domains from fungal NRPSs with known function are color coded: dark blue = peptaibol synthetases, light blue = ChNPS4 (clades grouping with each A domain of C. heterostrophus NPS4), green = AMT synthetases and ChNPS1 and ChNPS3 modules 1 and 3, orange = ergot alkaloid synthetases, light green = ChNPS8/PerA synthetases, and red = homologs of ChNPS6 (extracellular siderophore synthetases). Of these groups, only the peptaibol synthetases, the clade containing NPS8/PerA/NPS6 synthetases (arrow), and ChNPS4 modules 3 and 4 have bootstrap support >70%. C. heterostrophus NRPS A domains are indicated (circles).

Figure 8

Modular organization of Peptaibol synthetases and proposed evolution by tandem duplication. A domains from peptaibol synthetases form three distinct, well-supported clades in the EAS subfamily (Fig. 7). A. Modular structure of the H. virens TEX 1 peptaibol synthetase and two peptaibol synthetases in the related species, T. reesii (T.reesii 2_23171 and T.reesii 2_123786. Color coding corresponds to clades identified in phylogenetic analyses (B and C, and Fig. 7). Arrows indicate bootstrap support for module relationships (B, C. and Fig. 7). While T. reesii 2_23171 is clearly a homolog of TEX1, domain duplication of modules 18 to 19 or vice versa and addition of module 2 have occurred since the common ancestor of these species. B. Two of the peptaibol synthetases clades (light green and dark blue, Fig. 7), group together as a monophyletic group but without bootstrap support. A domains shown in stippled boxes indicate modules from T.reesii 2_123786 which do not have a clear counterpart in the other peptaibol synthetases and may represent ancestral domains. C. The third clade (purple, Fig. 7) groups in a distinct position within the EAS subtree.

Figure 9

Phylogenetic groupings and modular organization of ChNPS1 and ChNPS3 showing recombinant structure of these NRPSs. A. Modules 1 and 3 of both ChNPS1 and ChNPS3 group with AM toxin synthetase, a trimodular NRPS that biosynthesizes AM-toxin, an Alternaria alternata host-selective toxin. B. Module 2 of ChNPS1 and modules 2 and 4 of ChNPS3 group with A domains (SimA) of cyclosporin synthetases (CYCLO) in a disparate position in the larger phylogeny compared to modules 1 and 3 (A, above) which group in the EAS subfamily (Fig. 2). C. Recombinant domain organization of ChNPS1 and ChNPS3. Blue boxes correspond to modules 2 and 4, purple boxes to modules 1 and 3. Note that single modules homologous to these domains are found in other euascomycete NRPSs. For example, Enniatin synthetases (Esyn1) and MGG00022.6 are also recombinant like ChNPS1 and ChNPS3 with one or more modules grouping with the cyclosporin subfamily (blue boxes) and others also within the EAS subfamily but in a distinct position from the ChNPS1 and ChNPS3 modules (clear boxes). Cyclosporin synthetases itself appears to have arisen by tandem duplication of SimA modules within T. inflatum.

Figure 10

Number and range of NRPSs and A domains for each subfamily. A. Average and range (lowest to highest) number of NRPS-encoding genes in each subfamily per euascomycete genome shows that the EAS subfamily has both the highest average number of genes and the highest variation in copy number among species. PKS;NRPSs and ChNPS12 subfamilies also have substantial variation in numbers of NRPS-encoding genes among species. B. Average and range (lowest to highest) of the number of A domains/NRPS in euascomycete genomes for each subfamily shows that the EAS subfamily also has by far the greatest variation in number of A domains/NRPS followed by the CYCLO, and SID subfamilies.