A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

Abstract

Background: Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS). Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454") and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides.

Results: Thirteen types of oligopeptides were uncovered by mass spectrometry (MS) analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded) precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island.

Conclusion: Altogether seven nonribosomal peptide synthetase (NRPS) gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully can identify NRPS gene clusters and the corresponding oligopeptides. The analyses suggest independent evolution of all NRPS gene clusters as functional units. Our data indicate that the Planktothrix genome displays evolution of dual pathways (NRPS and ribosomal) for production of oligopeptides in order to maximize the diversity of oligopeptides with similar but functional discrete bioactivities.

Figure 1

Organization of the NRPS gene clusters found in the Planktothrix NIVA CYA 98 genome. Gene clusters shown are: microcystin [EMBL: AM990462], cyanopeptolin, anabaenopeptin and microviridin (putative ribosomal produced oligopeptide) on one contig [EMBL: AM990463], aeruginosin [EMBL: AM990465], microginin [EMBL: AM990464] synthetase gene clusters and two gene clusters with unassigned oligopeptides [EMBL: AM990466 and AM990467]. Contigs from the assembly of 454 reads are illustrated with black lines and gaps closed with Sanger sequencing are illustrated with red lines. Gene names, transcription directions and approximate sizes are illustrated with yellow boxes with arrows. Adenylation- (red; showing in silico predicted activated amino acids), condensation- (green), thiolation-(yellow), methyltransferase- (blue), epimerase- (turquoise) glyceric acid- (lilac), halogenation- (purple) and termination domains (grey) are shown by their first letter abbreviations. Polyketide domains are shown as circles. The ABC transporter genes (light green) are connected to six of the seven gene clusters.

Figure 2

Proposed microviridin and oscillatorin biosynthetic pathways. Orange arrows, with protein name and predicted function above, illustrate the genes size and transcription direction. a) MdnA is proposed as microviridin precursor. MdnCB are predicted to ligate the ring structures, MdnD is responsible for acetylation. A microviridin with unknown side chain was predicted based on the precursor sequence (MdnA) and gene similarities to the Microcystis microviridin gene clusters [16]. b) The alignment of sequences with high similarities to Microcystis MdnA shows two highly conserved regions in the leader peptide (green dashed line) and core peptide (red dashed line). The leader-core transition is unknown. c) oscA is proposed as the oscillatorin precursor encoding 619 amino acids, includes eight of the 10 amino acids in oscillatorin (bold text). The tryptophan is suggested as a precursor to oscillatoric acid (Osc). Based on in silico analyses of the gene cluster, the oscillatorin precursor is likely to be cleaved by peptidase S33. Modification of tryptophan and addition of valine is carried out by unknown enzymes. A putative cyclo-ligase is may be involved in cyclization.

Figure 3

ABC transporter tree constructed using Bayesian inference. Support values for the nodes are Bayesian posterior probability and NJ bootstrap replicates above 50%. Genus origin is denoted for taxa other than Planktothrix. Accession numbers for the sequences are listed in Additional file 3: Table S3. The CpRev substitution model and gamma-shaped distribution were used in the Bayesian analyses. The MCMC chains were carried out for 4 million generations with sampling of trees every 100 generations, removing the first 3000 trees.