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Supplementary Information: Additional file 1: Supplementary Figure S1. Photorhabdus luminescens incubated with the KC-12 probe during 70 h. Supplementary Note 1. Taxonomic Appendix. Supplementary Figure S2. Hybridization of cells from nudibranch skin, gut, gills and gonads. Supplementary Figure S3. KEGG modules of Ca. D. californiensis and medium quality Ca. Tethybacterales MAGs from sponges. Supplementary Figure S4. Sulfur and nitrogen metabolism of Ca. D. californiensis and medium quality Ca. Tethybacterales MAGs from sponges. Supplementary Figure S5. ABC transporters of Ca. D. californiensis and medium quality Ca. Tethybacterales MAGs from sponges. Supplementary Figure S6. Phylogenetic tree of 16S rDNA gene sequences from public datasets. Supplementary Figure S7. Proportion of reads with >92% sequence similarity to Ca. D. californiensis in other nudibranchs. Supplementary Figure S8. Heatmap showing relative abundances of top ASVs across all 16S rDNA amplicon samples. Supplementary Figure S9. Core microbiome of D. fulva mantle. Supplementary Figure S10. ASVs determining the ordination of samples in the PCA anlaysis of nudibranch microbiome samples. Supplementary Figure S11. Phylogenetic tree of full length 16S rDNA gene sequences from nudibranch skin and mucus obtained by Sanger sequencing. Supplementary Figure S12. Ca. D. californiensis betalactone gene cluster compared to the best antiSMASH matches. Supplementary Figure S13. Similarity of the genes in the Ca. D. californiensis betalactone gene cluster with the 'nr' database of NCBI. Supplementary Figure S14. Similarity of the genes in the Ca. D. californiensis betalactone gene cluster with other members of the Ca. Tethybacterales order. Supplementary Figure S15. Biosynthetic pathway comparison to known betalactone natural product pathways. Supplementary Figure S16. NMR analysis of extracts from D. fulva nudibranchs. Supplementary Figure S17. Quantification of the dcbD expression in the CRAGE strains and nudibran

[Background] Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva., [Results] We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously., [Conclusions] Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.

Supplementary data to this article: Supplementary Document 1. Detailed description of the databases construction. Supplementary Figure 1. Processing and construction workflow of reference databases. The final databases used in the comparison are highlighted in red. Supplementary Figure 2. Determination of the optimal number of clusters for average abundance clustering analysis. The Elbow method was used to set the optimal number of clusters for the k-means algorithm at genus (A) and species (B) level. Supplementary Figure 3. Determination of the optimal number of clusters for z-scored average abundance patterns analysis. The Elbow method was used to set the optimal number of clusters for the k-means algorithm at genus (A) and species (B) level. Supplementary Figure 4. Bracken results comparison between the enriched filtered-MAG and original NT databases. From top to bottom, the performance of the databases was examined in terms of classification capacity for all samples as a whole, by host lifestyles and geographic origin at genus (A) and species (B) level. In the box plots, the black line within the box marks the median and the red triangle the mean, outliers are presented as red dots. Supplementary Figure 5. Relative abundances of top gut microbiota at the genus level. Only the 25 most abundant taxa in all samples were represented. Taxa are sorted by average relative abundances and their NCBI’s taxID is indicated in parentheses. In the box plots, the black line within the box marks the median and the red triangle the mean. Supplementary Figure 6. Universal core species. (A) Intersections between cores of interest. (B) Taxonomic relationships between universal core species. The number of core taxa assigned to a particular level is indicated inside the square brackets. (C) Prevalence-Abundance Heatmap. Taxa are sorted by average relative abundances and their NCBI’s taxID is indicated in parentheses. Supplementary Figure 7. Taxonomic relationships between different i

The human gut holds a special place in the study of different microbial environments due to growing evidence that the gut microbiota is related to host health. However, despite extensive research, there is still a lack of knowledge about the core taxa forming the gut microbiota and, moreover, available information is biased towards western microbiomes in both genome databases and most core taxa studies. To tackle these limitations, we tested a database enrichment strategy and analyzed public datasets of whole-genome shotgun data, generated from 545 fecal samples, comprising three gradients of westernization. The NT database was selected as a baseline of biological diversity, subsequently being combined with various studies of interest related to the human microbiota. This enrichment strategy made it possible to improve classification capacity, compared to the original unenriched database, regarding the various lifestyles and populations studied. The effects of incomplete-taxonomy metagenome-assembled genomes on genome database enrichment were also examined, revealing that, while they are helpful, they should be used with caution depending on the taxonomic level of interest. Moreover, in terms of high prevalence, the core analysis revealed a conserved set of bacterial taxa in the healthy human gut microbiota worldwide, despite apparent lifestyle differences. Such taxa show a set of traits, metabolic roles, and ancestral status, making them suitable candidates for a hypothetical phylogenetic core of mutualistic microorganisms co-evolving with the human species.

Our study is a pioneering exploration of the microbiome in the soil of the Sumaco stratovolcano and an assessment of the effects of an elevational gradient and related physicochemical soil parameters on richness and community structure. The Sumaco, as an isolated Amazonian stratovolcano, may be among one of the least studied ecosystems in Ecuador and perhaps the Amazon region. Universal patterns remain unresolved or available information inconclusive to establish a supported consensus on general governing processes by which elevation and its associated environmental gradients may determine the microbial richness and community structure. We tested a recent proposal on how microbial diversity responds to montane gradients, placing a central role in soils as potentially independent of altitude along an elevational gradient. Correlations and effects among soil physicochemical parameters and altitude were contrasted against richness and community structure through quantitative ecology. The most informative physicochemical parameter in our assessment of bacterial community structure was neither pH nor altitude, but sulfur, which was mostly independent of the other tested parameters. We established a positive effect of richness by parameters associated with metallic cations such as Mn2+, and CEC, which were negatively correlated to altitude and pH. The possible relation between the significant role of sulfur on bacterial community structure with the unique geological origin of the Sumaco stratovolcano should be examined in the context of specialized sulfur metabolisms and additional information on community structure and environmental constraints. Our study establishes an initial baseline for further explorations of microbial diversity in this unexplored tropical stratovolcano.

All metagenomics classifiers require a pre-computed database of reference microbial genomes. Among them for shotgun metagenomics the most popular reference databases are the BLAST nucleotide collection (NT Database) and the Reference Sequence Database (RefSeq Database) for high-quality nucleotide sequences. Despite their importance, there are no available comparisons in terms of classification performance between them. In this study we focus on the case of the human gut microbiota to carry out a benchmarking of different conventional reference genome databases (including NT Database and RefSeq Database at Complete Genomes and Complete Genomes + Chromosome assembly levels) and evaluate their performance using simulated and experimental datasets. These results intend to provide some insights when choosing reference genome databases in a case of special interest such as the study of the human gut microbiota.