Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

Abstract

Background: Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simultaneously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis.

Results: Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.

Conclusion: Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

Figure 1

The highly toxic microcystin-LR variant. The microcystin chemical structure can be generalized as cyclo-D-Ala¹-X²-D-MeAsp³-Z⁴-Adda⁵-D-Glu⁶-Mdha⁷ where X and Z denote the highly variable second and fourth positions. Microcystins may contain L-Ala, L-Arg, L-Glu, L-Hil, L-Hph, L-Hty, L-Leu, L-Met, L-Phe, L-Try, L-Tyr, or L-Val at the X position and L-Aba, L-Ala, L-Arg, L-Glu, L-Har, L-Leu, L-Met, L-Phe, L-Try, or L-Tyr at the Z position [1].

Figure 2

The modular organization of McyA, McyB and McyC proteins. These three proteins catalyze 6 rounds of elongation and the final cyclisation of the heptapeptidyl microcystin intermediate. The McyB1 and McyC adenylation domains are responsible for the recognition and activation of the amino acids found at the X and Z positions in the microcystin respectively [11]. The McyB1 and McyC condensation domains are responsible for peptide bond formation between this activated amino acid and the growing peptide chain [11]. Each circle represents a NRPS enzymatic domain: A, aminoacyl adenylation; M, N-methyltransferase; T, Thiolation domain, C, condensation; E, epimerization; Te, thioesterase.

Figure 3

The relative proportions of amino acids incorporated into the X and Z position. The relative proportions of amino acids incorporated into the X and Z position of the microcystins produced by the strains included in this study as determined by LC-MS. (a) The amino acids present at the X position in microcystins. These amino acids are recognized and activated by the McyB1 adenylation domain. (b) The amino acids present at the Z position in microcystins. These amino acids are recognized and activated by the McyC adenylation domain. The structures and percentages of individual microcystins produced by the 10 strains of cyanobacteria included in this study are given in the supplementary information section (see additional file 1).

Figure 4

A phylogenetic compatibility matrix of mcyB1 and mcyC genes from 10 strains of toxic cyanobacteria. A phylogenetic compatibility matrix of mcyB1 and mcyC genes from 10 strains of toxic cyanobacteria. The matrix was constructed through comparing congruence between subtrees of whole alignment. At first, 67 alignment fragments were obtained by moving a 300 nucleotide window along the alignment with a step of 50 bases, and neighbor-joining tree of each fragment was constructed by PHYLIP. Then phylogenetic violations of any two different subtrees were calculated by TREEORDERSCAN (Simmonic 2005 version 1.5), and proportionally presented as a colour gradient showed in the figure. The NRPS enzymatic domains present in McyB1 and McyC are indicated: A, aminoacyl adenylation; C, condensation; T, Thiolation domain; Te, Thioesterase.

Figure 5

Breakpoints density plot along the alignment of mcyB and mcyC genes. Light grey and dark grey areas respectively indicate local 99% and 95% breakpoint clustering thresholds taking into account regional differences in sequence diversity that influence the ability of different methods to detect recombination breakpoints. The broken line in the breakpoint density graph indicate 95% confidence thresholds for globally significant breakpoint clusters. The boundary between the condensation, adenylation and thiolation domains is indicated with a solid line.

Figure 6

Phylogenetic congruence between housekeeping and microcystin synthetase genes. Congruence between housekeeping genes of the producer organism (16S rRNA, rpoC1, rpoB, tufA and rbcL) on the left and the microcystin synthetase genes (mcyD, mcyE, and mcyG) on the right. Maximum-likelihood tree based on five housekeeping genes (-lnL = 20872.57747) and 3 microcystin synthetase genes (-lnL 21445.80119). Bootstrap values above 50% from 1000 maximum-likelihood bootstrap replicates are given at the nodes. Branch lengths are proportional to sequence.

Figure 7

Discordant phylogenetic relationships between the McyB1 and McyC condensation and adenylation domains. (a) A maximum-likelihood tree based on the McyB1 and McyC condensation domains (C) reflecting separate evolutionary history for these two condensation domains indicating the chirality of the amino acid at the donor site of the condensation domain (-lnL = 21740.48206). (b) A maximum-likelihood tree based on the McyB1 and McyC adenylation domains (A), from A3–A8, showing intermixed cluster of McyB1 and McyC adenylation reflecting a more recent evolutionary history characterized by periods of replacement through recombination leading to domain replacement (-lnL = 10308.24462).