Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Gut microbiomes of cycad-feeding insects tolerant to ß-methylamino-L-alanine (BMAA) are rich in siderophore biosynthesis.

Ingestion of the cycad toxins ß-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of selected bacteria. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate selected taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We characterized the biosynthetic potential of four bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages, and discovered a suite of biosynthetic gene clusters notably rich in siderophores. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, including desferrioxamine B, suggesting the occurrence of an unprecedented desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, including a possible role for bacterial siderophores.

Metagenomics Bioinformatic Pipeline.

Microbial communities' taxonomic and functional diversity has been broadly studied since sequencing technologies enabled faster and cheaper data obtainment. Nevertheless, the programming skills needed and the amount of software available may be overwhelming to someone trying to analyze these data. Here, we present a comprehensive and straightforward pipeline that takes shotgun metagenomics data through the needed steps to obtain valuable results. The raw data goes through a quality control process, metagenomic assembly, binning (the obtention of single genomes from a metagenome), taxonomic assignment, and taxonomic diversity analysis and visualization.

Evolutionary Genome Mining for the Discovery and Engineering of Natural Product Biosynthesis.

Genome mining has become an invaluable tool in natural products research to quickly identify and characterize the biosynthetic pathways that assemble secondary or specialized metabolites. Recently, evolutionary principles have been incorporated into genome mining strategies in an effort to better assess and prioritize novelty and understand their chemical diversification for engineering purposes. Here, we provide an introduction to the principles underlying evolutionary genome mining, including bioinformatic strategies and natural product biosynthetic databases. We introduce workflows for traditional genome mining, focusing on the popular pipeline antiSMASH, and methods to predict enzyme substrate specificity from genomic information. We then provide an in-depth discussion of evolutionary genome mining workflows, including EvoMining, CORASON, ARTS, and others, as adopted by our group for the discovery and prioritization of natural products biosynthetic gene clusters and their products.

Cycad Coralloid Roots Contain Bacterial Communities Including Cyanobacteria and Caulobacter spp. That Encode Niche-Specific Biosynthetic Gene Clusters.

Cycads are the only early seed plants that have evolved a specialized root to host endophytic bacteria that fix nitrogen. To provide evolutionary and functional insights into this million-year old symbiosis, we investigate endophytic bacterial sub-communities isolated from coralloid roots of species from Dioon (Zamiaceae) sampled from their natural habitats. We employed a sub-community co-culture experimental strategy to reveal both predominant and rare bacteria, which were characterized using phylogenomics and detailed metabolic annotation. Diazotrophic plant endophytes, including Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc species, dominated the epiphyte-free sub-communities. Draft genomes of six cyanobacteria species were obtained after shotgun metagenomics of selected sub-communities. These data were used for whole-genome inferences that suggest two Dioon-specific monophyletic groups, and a level of specialization characteristic of co-evolved symbiotic relationships. Furthermore, the genomes of these cyanobacteria were found to encode unique biosynthetic gene clusters, predicted to direct the synthesis of specialized metabolites, mainly involving peptides. After combining genome mining with detection of pigment emissions using multiphoton excitation fluorescence microscopy, we also show that Caulobacter species co-exist with cyanobacteria, and may interact with them by means of a novel indigoidine-like specialized metabolite. We provide an unprecedented view of the composition of the cycad coralloid root, including phylogenetic and functional patterns mediated by specialized metabolites that may be important for the evolution of ancient symbiotic adaptations.