Convergent evolution of [D-Leucine(1)] microcystin-LR in taxonomically disparate cyanobacteria

Abstract

Background: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly.

Results: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu(1)] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA(2) adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA(2) adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu(1)] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met(1)] microcystin-LR.

Conclusions: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu(1)] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Figure 1

Microcystin chemical structures and biosynthetic enzymes. (a) [D-Leu¹] microcystin-LR and (b) [D-Met¹] microcystin-LR. MeAsp: D-erythro-β-methylaspartic acid. Adda: (2S,3S,8S,9S)-3-,amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. Mdha: N-methyldehydroalanine. X and Z are the highly variable positions. (c) NRPS and PKS involved in microcystin synthesis from Microcystis. The order of the enzymes corresponds to the assembly of microcystin (each position of the microcystin structure is indicated under each adenylation domain). N. N-methyltransferase, Ep. epimerization, TE. thioesterase domains are shown in white and the PCP. peptidyl carrier protein domain in black. Regions included in the phylogenetic analysis are indicated in grey, in addition of their respective PCP domains.

Figure 2

Photomicrographs of the studied strains. (a) Microcystis aeruginosa NPLJ-4. (b) Microcystis sp. RST 9501. (c) Nostoc sp. UK89IIa. (d) Phormidium sp. CENA270.

Figure 3

Phylogenetic analysis of the 16S rRNA gene. Maximum-likelihood tree based on the 16S rRNA gene. Bootstrap values above 50% from 1000 maximum-likelihood bootstrap replicates are given at the nodes. The studied strains are in bold and highlighted. Symbols for microcystin variants in position one: ● [D-Ala¹]; ■ [D-Leu¹]; ★ [D-Met¹]; ◣ [D-Ser¹].

Figure 4

Conservative evolutionary history of domains surrounding the McyA₂adenylation domain in the microcystin biosynthetic gene cluster. Maximum-likelihood tree based on amino acid sequences of the (a) condensation domain, (b) peptidyl carrier protein domain and (c) epimerization domain within the mcy gene cluster. Phylogenetic tree inferred using MEGA 5. Bootstrap values above 50 per cent from 1000 respectively neighbor-joining, maximum parsimony and maximum-likelihood bootstrap replicates are given at the nodes. The studied strains are in bold and indicated with *.

Figure 5

Evolutionary history of adenylation domains of microcystin biosynthetic gene cluster. Maximum-likelihood tree based on amino acids sequences of adenylation domain within mcy gene cluster. Phylogenetic tree inferred using MEGA 5. Bootstrap values above 50 per cent from 1000 respectively neighbor-joining, maximum parsimony and maximum-likelihood bootstrap replicates are given at the nodes. The studied strains are in bold and indicated with *.

Figure 6

Independent evolutionary history of McyA₂ adenylation domain. Maximum-likelihood tree based on amino acids sequences inferred using MEGA 5. Bootstrap values above 50 per cent from 1000 respectively neighbor-joining, maximum parsimony and maximum-likelihood bootstrap replicates are given at the nodes. The studied strains are in bold and indicated with *.

Figure 7

Recombination breakpoints in the mcyA₂ gene detected by the RDP3 program. (a) Breakpoint positions detected in three cyanobacterial strains. The position in the alignment is in parentheses. *The breakpoint position is undetermined. (b) Potential recombination hotspots within the alignment of mcyA₂ genes. The black line corresponds to the breakpoint count within a moving 200-nt-long window. White and grey areas respectively indicate local 99% and 95% confidence intervals of the hot/cold spot test. Upper and lower broken lines respectively indicate 99% and 95% confidence thresholds for the global hotspot test.