Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains

Abstract

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.

FIG. 1.

General structure of microcystin. The general structure of microcystin is cyclo(d-Ala-l-X-d-MeAsp-l-Z-Adda-d-Glu-Mdha-), where X and Z are variable l amino acids, Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, d-MeAsp is d-erythro-β-methyl-aspartic acid, and Mdha is N-methyl-dehydroalanine. In the most common variant (MC-LR) X is leucine (L) and Z is arginine (R).

FIG. 2.

Organization of the mcyABC gene cluster in strain PCC 7806. The module arrangement and HindIII restriction sites are shown. In the diagram at the bottom, the relative positions of primers (arrows) and the Southern probe are shown. Abbreviations: H, HindIII restriction sites; s, spacer (His motif); A1, adenylation core motif 1; T, thiolation core motif. For simplicity, condensation core motifs are not shown.

FIG. 3.

Southern blot analysis of the first module in mcyB. Genomic DNA was digested with HindIII and hybridized with an mcyB probe (Fig. 2) (A) and a 16S rDNA probe (B). The 16S rDNA hybridization results are shown for loading comparisons.

FIG. 4.

PCR of two regions in the mcyABC operon. Standard PCR was performed with regions mcyAB (primers 135-F and 676-R) (A) and mcyB (primers 2156-F and 3111-R) (B) (Fig. 2). The marker used was φX174/HaeIII.

FIG. 5.

Alignment of the amino acid sequences of region 2156-3111 from the 12 strains possessing the mcyB gene and the corresponding sequences in the mcyC gene from strains PCC 7806 and K-139 (PCC 7806c and K-139c, respectively) and the grsA gene from Bacillus brevis. Red, polar, charged amino acids; orange, polar, uncharged amino acids; light green, hydrophobic amino acids; dark green, small amino acids. The nine amino acids putatively involved in substrate recognition are indicated by arrows, core sequences are underlined, and genetic variants are indicated at the end of the alignment.

FIG. 6.

Phylogenetic and UPGMA analyses. Phylogenetic analyses were performed with both nucleotide (A) and amino acid (B) alignments from the region amplified by primers 2156-F and 3111-R. The corresponding region in grsA (accession no. M29703) from B. brevis was used as an outgroup. (A) Tree constructed by the minimum-evolution method based on a distance matrix computed by the Kimura two-parameter model. (B) Neighbor-joining tree constructed based on distance measurements estimated by the Dayhoff PAM matrix. Bootstrap values (in percentages) from 500 replicates are indicated at the branch nodes. (C) UPGMA analysis of both the RAPD and REP matrices based on Jaccard similarities between strains. The arrows point towards the positions of the same strains in the mcyB1 (nucleotide) phylogenetic tree. Abbreviations: PCC 7806c, mcyC module from PCC 7806; K-139c, mcyC module from K-139; aer, M. aeruginosa; vir, M. viridis; sp, Microcystis sp.; bot, M. botrys; fl-a, Microcystis cf. flos-aquae; icht, Microcystis cf. ichthyoblabe; wes, Microcystis cf. wesenbergii.

FIG. 7.

RT-PCR for region mcyAB. A standard RT-PCR was performed with the gene-specific primers 135-F and 676-R. There is a weak signal in lane N-C 169/7.