Comparison of cyanopeptolin genes in Planktothrix, Microcystis, and Anabaena strains: evidence for independent evolution within each genus

Abstract

The major cyclic peptide cyanopeptolin 1138, produced by Planktothrix strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb oci gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from Anabaena and Microcystis, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the Planktothrix and Microcystis cyanopeptolin domains than between these and the Anabaena domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.

FIG. 1.

Putative peptide structure of cyanopeptolin 1138 produced by Planktothrix strain NIVA CYA 116. Asterisks denote that the chemical analysis cannot distinguish between Ile, Leu, and allo-Ile. However, in silico analyses clearly suggest Ile at positions 5 and 7.

FIG. 2.

Architectures of the oci gene cluster from Planktothrix (GenBank accession no. DQ837301), mcn from Microcystis (GenBank accession no. DQ075244), and apd from Anabaena (GenBank accession no. AJ269505), all encoding cyanopeptolin synthetases. Gene names, transcription directions, and approximate sizes are shown with yellow arrows. A (with putative activated amino acids) (red), C (green), T (yellow), methyltransferase (blue), sulfotransferase (pink), halogenation (purple), and termination (gray) domains are shown with their first-letter abbreviations. Corresponding modules are shown alternately in light gray or light blue. The ABC transporter gene is located upstream of the synthetase genes, is transcribed in opposite directions in the oci and mcn gene clusters, and appears downstream of the apd gene cluster.

FIG. 3.

(A) A-domain tree constructed using Bayesian inference, where domains group by function (i.e., activated amino acid). Support values for the nodes are Bayesian posterior probability (in bold) and 1,000 bootstrap replicates (in regular font) deduced from the NJ tree with a nearly identical structure (with a minor topology difference within Microcystis group III). Only values above 50% are shown. Genus origin is denoted with first-letter abbreviations (P, Planktothrix; M, Microcystis; A, Anabaena; and N, Nostoc). The putative activated amino acids are also shown (Ad, d-alanine; Hty, Htyr; Ed, d-glutamate; Dm, methylaspartic acid). The WAG substitution model was used in the Bayesian analyses. (B) A-domain split tree constructed using SplitsTree4 at the default setting, showing 1,000 bootstrap replicas above 50%. To investigate recombinations between cyanopeptolin gene clusters, microcystin domains were removed.

FIG. 4.

(A) C-domain tree constructed using Bayesian inference, where domains group mainly by function (homologous C domains). Bayesian posterior probability and bootstrap values (from the NJ tree with an identical structure) above 50% are shown. The CpRev protein substitution model was used in the Bayesian analyses. Genus origins are shown with first-letter abbreviations (Fig. 3), and the C domains are labeled in numerical order according to transcription direction (i.e., seven oci, seven mcn, and six apd C domains). (B) C-domain split tree constructed using SplitsTree4 at the default setting, showing 1,000 bootstrap replicas above 50%. To investigate recombinations between cyanopeptolin gene clusters, microcystin domains were removed.

FIG. 5.

ABC transporter tree constructed using Bayesian inference, where the ABC transporters group according to associated NRPS family; deep branches, however, are unresolved. Postprobability values above 50% are shown. Bayesian analyses were performed with the CpRev protein substitution model and gamma distribution to account for variable substitution rates across sites. Uncharacterized ABC transporters are identified with the following GeneInfo Identifier numbers: for Crocospaera, gi67921872; for Nostoc ATCC 29133, gi23129070; and for Nostoc PCC 73102, gi23128856.