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Distribution and Evolution of Nonribosomal Peptide Synthetase Gene Clusters in the Ceratocystidaceae.

ABSTRACT: In filamentous fungi, genes in secondary metabolite biosynthetic pathways are generally clustered. In the case of those pathways involved in nonribosomal peptide production, a nonribosomal peptide synthetase (NRPS) gene is commonly found as a main element of the cluster. Large multifunctional enzymes are encoded by members of this gene family that produce a broad spectrum of bioactive compounds. In this research, we applied genome-based identification of nonribosomal peptide biosynthetic gene clusters in the family Ceratocystidaceae. For this purpose, we used the whole genome sequences of species from the genera Ceratocystis, Davidsoniella, Thielaviopsis, Endoconidiophora, Bretziella, Huntiella, and Ambrosiella. To identify and characterize the clusters, different bioinformatics and phylogenetic approaches, as well as PCR-based methods were used. In all genomes studied, two highly conserved NRPS genes (one monomodular and one multimodular) were identified and their potential products were predicted to be siderophores. Expression analysis of two Huntiella species (H. moniliformis and H. omanensis) confirmed the accuracy of the annotations and proved that the genes in both clusters are expressed. Furthermore, a phylogenetic analysis showed that both NRPS genes of the Ceratocystidaceae formed distinct and well supported clades in their respective phylograms, where they grouped with other known NRPSs involved in siderophore production. Overall, these findings improve our understanding of the diversity and evolution of NRPS biosynthetic pathways in the family Ceratocystidaceae.

SUBMITTER: Sayari M

PROVIDER: S-EPMC6563098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S-23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.

Project description: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.

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Distribution and Evolution of Nonribosomal Peptide Synthetase Gene Clusters in the Ceratocystidaceae.

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