Identification of the rare [D-Leu1]MC and other microcystin variants
Microcystins form a large family of cyclic toxins characterized by a highly conserved chemical structure with an extensive amino acid composition at the two variable positions, two and four (Additional file
1: Figure S7 and Table S3). In this study, we detected rare [D-Leu1]MC variants from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Almost all microcystins reported in the literature contain D-Ala in position 1
. Previously, microcystins containing D-Leu
, and D-Ser
[36, 37] have also been reported. The [D-Leu1]MC variant has previously been found from Microcystis aeruginosa NPLJ-4, Microcystis sp. RST 9501, and water blooms from Brazil and Canada dominated by Microcystis strains
[27–31, 34]. The production of microcystins in lichen thalli by Nostoc symbionts has previously been reported
[37, 38]. Nostoc strains isolated from lichen symbiosis produce a large variety of microcystins including the [D-Leu1]MC variant
Microcystins are best known from aquatic habitats, where they are frequently reported from blooms. Although microcystins are more commonly detected in planktonic strains, terrestrial and benthic strains have also been reported to be producers of these compounds
[8, 39]. Benthic environmental samples containing microcystins have been reported from Switzerland
, and Antarctica
. Microcystin production in isolated cyanobacterial strains from benthic environments has been reported from Egypt
, New Zealand
, and the USA
. It is not always clear which cyanobacterium produces the toxin in benthic mats of cyanobacteria. A strain of the genus Phormidium was isolated from the walls of a reservoir in the USA and shown to produce a range of microcystin variants, all of which contained D-Ala (Additional file
1: Table S3)
. Our results demonstrated that Phormidium sp. CENA 270, isolated from a pond in the northeast of Brazil, also produces microcystins but with D-Leu in place of D-Ala. In the phylogenetic analysis of the 16S rRNA gene, the two Phormidium strains cluster together with Lyngbya, Oscillatoria, Phormidium and Oscillatoriales (Additional file
1: Figure S1). The biomass of benthic strains can go unnoticed to the casual observer but be massive enough to cause animal poisonings
[40, 45]. Moreover, when the strains lyse, microcystins are released into the water, which suggests that the analysis of toxic benthic cyanobacteria is also important in water-quality management.
The chemical structure of microcystins is highly conserved, with variation at X and Z positions (Figure
1a and b) resulting in over 86 reported variants
. In this study, we demonstrated that Microcystis strains NPLJ-4 and RST 9501 produce new microcystin variants containing methionine in addition to the rare [D-Leu1]MC variants. Met is also present in oscillamide B
 and in microcystin-M(O)R and -YM
. Nodularia spumigena strains also produce nodulapeptins, which commonly contain Met
[49, 50]. According to analysis using the NRPS Norine database, Met contains a methylthiol group and is rare in non-ribosomal peptides
. The presence of the highly active sulfhydryl group in the thiol group could explain the scarcity of secondary metabolites containing Met or Cys. If amino acid recognition by the McyA2 adenylation domain is not strict, the incorporation of Met instead of Leu is logical because of the similar size and hydrophobicity of the side chains. Here, we demonstrated that methionine is incorporated in the microcystins produced by Microcystis strains from brackish water. Microcystin variants are constantly being discovered, making the microcystin family extremely diverse, and posing a challenge for the detection of microcystins from water samples.
Convergence on [D-Leu1]MC variant chemical structure
Phylogenetic analysis of the McyA2 adenylation domains provided evidence for independent evolutionary events affecting the substrate specificity of the enzyme in three disparate genera of cyanobacteria (Figures
6). Breakpoint analysis suggests the replacement of almost the entire substrate specificity-conferring portion of the adenylation domain in Phormidium sp. CENA270 and Nostoc sp. UK89IIa. These gene conversions dramatically altered the predicted substrate specificity of the McyA2 adenylation domain in these strains and are linked to the synthesis of the [D-Leu1]MC variant. However, point mutations affecting the substrate specificity of the McyA2 adenylation domain in Microcystis strains NPLJ-4 and RST 9501 led to the synthesis of the [D-Leu1]MC and [Met1]MC variants.
Phormidium sp. CENA270 and Nostoc sp. UK89IIa are not grouped together with other McyA2 adenylation domains (Figure
6). The McyA2 adenylation domain of Phormidium sp. CENA270 grouped with the McyB1 adenylation domain from Microcystis strains, which produce microcystin variants containing D-Ala
[10, 52]. The adenylation domains of NosA1 and NosC1 from the nostopeptolide gene cluster are placed in the same clade with the McyA2 adenylation domain from Nostoc sp. UK89IIa (Figure
6). They are involved in the incorporation of Ile/Leu/Val and Leu, respectively, in Nostoc sp. GSV224
Genetic variation in the microcystin synthetases can be visualized in the phylogenetic trees showing two different patterns. While the amino acids of condensation, peptidyl carrier protein, and epimerization domain regions can be grouped according to the enzyme sequence (McyA, McyB, McyC, McyE, and McyG grouped together), the adenylation domain phylogeny clearly indicates recombination (Figures
5). Recombination in adenylation domains has previously been described for the adenylation and condensation domains of McyB1 and McyC
. The recombination and positive selection in the McyB1 adenylation domain are involved in the high variability of amino acids incorporated at position 2 of the microcystin
[16, 18, 22, 24, 25]. These genetic events have been related to the increase in the chemical diversity of microcystin. Interestingly, our results show that these different evolutionary events are involved in the convergence of the [D-Leu1]MC-LR.
Nevertheless, the selective forces behind this convergent evolution remain unclear. Competition in brackish water and different seasonal periods have possibly acted as selective forces. The chemical diversity of microcystins could be related to protein phosphatase inhibition as a form of chemical defense, for example against predators. Previous studies have indicated that microcystins can affect some predators, acting as metal chelators, in gene regulation, or in the inter- and intra-specific signaling
[54–59]. However, microcystins join a large number of secondary metabolites produced by different organisms that have no assigned biological function. According to the most accepted view, these compounds are produced due their ecological or physiological function and benefits for the producer organisms
. However, more information is still needed concerning the advantages in the production of these secondary metabolites. The biological role of a mixture of different bioactive compounds produced by the same strain would be interesting to study.
Prediction of McyA2 adenylation domain substrate specificity
The eight to ten amino acid residues forming the adenylation domain binding pocket are the main determinants of substrate specificity
[7, 61, 62]. In our study, Phormidium sp. CENA270 and Nostoc sp. UK89IIa were shown to produce [D-Leu1]MC variants and have identical binding pocket sequences (Table
2). Such amino acids signatures had been already described as presenting Leu specificity
. The Microcystis strains NPLJ-4 and RST 9501 differ in the binding pocket positions 301, 330, and 331 from the strains producing [D-Ala1]MCs. Residues at positions 301 and 330 are regarded to be less variable than at position 331
The adenylation domain binding pocket of Microcystis strains NPLJ-4 and RST 9501 has three different amino acid residues and a broader diversity of microcystin variants at position 1. Despite the fact that almost the entire binding pocket of Phormidium sp. CENA270 and Nostoc sp. UK89IIa differs from the other studied strains, only microcystin variants containing Leu at position 1 were detected. Re-engineering of non-ribosomal peptides has been a challenge in order to synthesize new peptides or to increase the activity of known compounds. The engineering of NRPSs to change substrate specificity can in some cases be achieved by point mutations. However, our results suggest that the replacement of entire domains might be a more successful strategy for producing a single product.
Replacement of almost the entire McyA2 adenylation domain in Nostoc sp. UK89IIa and Phormidium sp. CENA270 resulted in specificity towards Leu. Neither strain produced detectable levels of microcystin variants that contain other amino acids at this position. The recombination detected in the mcyA
gene of these strains affects the substrate-conferring portion of the McyA2 adenylation domain, which is important for the selection and activation of amino acids
[7, 61, 62]. Previously, it has been reported that recombination among different adenylation domains from mcyB1 and mcyC genes has led to a change in amino acid activation
We designed an experiment in order to test whether point mutations at positions 301, 330, and 331 could change the substrate specificity of the adenylation domain. However, single amino acids changes did not have the expected results. All the constructs and the wild type were found to activate valine in ATP-pyrophosphate (PPi) exchange assays. A previous study
 demonstrated that in the case of single or multiple mutations, the specificity of the wild type is not lost, but there is an increase in new substrate specificity. A comparison of adenylation domains from Microcystis strains that activate Ala and Leu reveals that several amino acid residues differ between them (Additional file
1: Figure S5). Of these different amino acid residues, five are 8 Å or less distant from the substrate and only three belong to the binding pocket. Although it is predicted that amino acid residues in the binding pocket are involved in selectivity, the catalytic efficiency could also be affected by the tertiary structure and proteinogenic surrounding area of the adenylation domain
. Promiscuity of the enzymes, allowing them to activate different substrates, could also be involved in the high variability of microcystin variants. Promiscuous activation of amino acids with a hydrophobic side chain by TycA, involved in the synthesis of the antibiotic tyrocidine A, has been reported
. Moreover, adenylation domains activating multiple substrates have been described from the fengycin
, and cyanopeptolin
 biosynthetic pathways.