Microcystin biosynthesis in planktothrix: genes, evolution, and manipulation

Abstract

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

FIG. 1.

Microcystin and nodularin isoforms. (For a review, see reference .) The structure of d-Asp-MCYST-RR is given as an example. Amino acids missing in the nodularin structure are indicated in red. The asterisk indicates the Mdha moiety that is replaced by Mdhb in the nodularin structure. Abbreviations: Aba, amino-isobutyric acid; Dha, dehydroalanine; Mdha, N-methyl-dehydroalanine; Met(O), methionine-S-oxide; Glu(OMe), glutamate methyl ester; (H₄)Y, 1,2,3,4-tetrahydrotyrosine; DMAdda, desmethyl-Adda; Dhb, dehydrobutyric acid; MeLan, N-methyl-lanthionin; ADMAdda, O-acetyl-Adda.

FIG. 2.

Organization of the gene clusters for microcystin biosynthesis in P. agardhii and M. aeruginosa. Genes coding for nonribosomal peptide synthetases or PKSs are indicated in light and dark gray, respectively. Hatched regions indicate genes with putative microcystin tailoring functions. Genes which are unique in each cluster are in boldface (33).

FIG. 3.

Domainwise comparison of microcystin synthetases from M. aeruginosa PCC 7806 and P. agardhii CYA 126. Colors indicate the degree of similarity.

FIG. 4.

Inactivation of the O-methyltransferase gene (mcyJ). (A) Schematic diagram of the deletional mutation of the mcyJ gene by homologous recombination. (B) PCR amplification with the DNA from the mutant (lane 1), wild type (lane 2), construct (lane 3), and negative control (lane 4) and the primer pair mcyJF and mcyISR. Lambda/PstI marker (lane 5) shows sizes in kilobases. The amplicon with mutant DNA is of the same size as that of the construct, which includes the chloramphenicol resistance marker (Cm^r).

FIG. 5.

MALDI-TOF MS analysis of whole cells from the wild type (upper spectrum) and the mcyJ mutant (lower spectrum). Major peaks in the wild type include [d-Asp]-MCYST-RR (m/z 1024), whereas this peak in the mutant is replaced by [DMAdda-d-Asp]-MCYST-RR (m/z 1010).

FIG. 6.

Alignment of the adenylation (A) domain core motifs (23) of microcystin synthetases from P. agardhii and M. aeruginosa (rows P.a. and M.a., respectively). The consensus sequences for motifs A1 to A10 of the peptide synthetase adenylation domains are indicated above. Amino acids in P. agardhii and M. aeruginosa that are identical to the consensus sequences are indicated in red. Boldface type indicates amino acids that are the same in P. agardhii and M. aeruginosa but differ from the consensus.

FIG. 7.

Comparison of the putative specificity-conferring codes of adenylation domains (3, 32) in microcystin biosynthesis from P. agardhii and M. aeruginosa. Residues conserved in both genera are indicated in boldface type (http://raynam.chm.jhu.edu/∼nrps/).