Background: For many environments, biome-specific microbial gene catalogues are being recovered using shotgun metagenomics followed by assembly and gene calling on the assembled contigs. The assembly is typically conducted either by individually assembling each sample or by co-assembling reads from all the samples. The co-assembly approach can potentially recover genes that display too low abundance to be assembled from individual samples. On the other hand, combining samples increases the risk of mixing data from closely related strains, which can hamper the assembly process. In this respect, assembly on individual samples followed by clustering of (near) identical genes is preferable. Thus, both approaches have potential pros and cons, but it remains to be evaluated which assembly strategy is most effective. Here, we have evaluated three assembly strategies for generating gene catalogues from metagenomes using a dataset of 124 samples from the Baltic Sea: (1) assembly on individual samples followed by clustering of the resulting genes, (2) co-assembly on all samples, and (3) mix assembly, combining individual and co-assembly. Results: The mix-assembly approach resulted in a more extensive nonredundant gene set than the other approaches and with more genes predicted to be complete and that could be functionally annotated.The mix assembly consists of 67 million genes (Baltic Sea gene set, BAGS) that have been functionally and taxonomically annotated. The majority of the BAGS genes are dissimilar (< 95% amino acid identity) to the Tara Oceans gene dataset, and hence, BAGS represents a valuable resource for brackish water research. Conclusion: The mix-assembly approach represents a feasible approach to increase the information obtained from metagenomic samples.

Biome-specific gene catalogues have been recovered for many environments using shotgun metagenomics, followed by assembly and gene calling on the assembled contigs. We recently proposed a novel mix-assembly strategy, combining individual and co-assembly approaches, and used this approach to assemble an extensive non-redundant gene set from 124 Baltic Sea metagenome samples. The Baltic Sea Gene Set (BAGS v1.1) comprises 66.53 million functionally and taxonomically annotated genes. To enable interactive exploration of this gene catalogue we have developed an RShiny application, BAGS-Shiny, that allows users to perform searches by sequence similarity (BLAST) and/or taxonomic and functional annotation. The gene catalogue and web application will serve as valuable tools for exploring microbial gene functions in brackish ecosystems. In addition, we here make available a pipeline to create gene catalogues based on the mix-assembly approach.

Vibrio vulnificus is a natural part of the microbiome of brackish waters worldwide. It is also an opportunistic pathogen that can cause severe infections and septicemia via consumption of seafood or through wound infections. The species possess diverse virulence factors, yet its precise disease mechanism remains undefined. Comparative genomics between clinical and environmental isolates offers a means to identify key virulence genes, but the scarcity of environmental isolates for V. vulnificus has constituted a significant limitation. Here we sequenced genomes of 82 V. vulnificus isolates from water, sediment and seagrass surface from stations along the Baltic Sea coast and complemented these with 208 and 117 previously sequenced clinical and environmental genomes, respectively, in a comparative analysis. Phylogenetic reconstruction corroborated earlier analysis with four main lineages forming within the species. Strains from the Baltic Sea region were confined to certain phylogenetic lineages (L4 and sublineages L2c and L2e) whereas clinical and environmental strains were found in all lineages, indicting that the phylogenetic structure of V. vulnificus reflects adaptations to specific environmental conditions rather than pathogenicity. Employing orthologue enrichment analysis in a phylogenetic framework using the PhyloBOTL pipeline developed in this work revealed 58 significantly enriched orthologs in clinical compared to environmental isolates. These orthologs were grouped into 18 co-localisation clusters based on the corresponding genes’ proximity in the genomes. The co-localisation clusters entailed clusters with 1 genes previously linked with pathogenicity in V. vulnificus, such as genes for capsular polysaccharide (CPS) synthesis and biofilm formation, but also clusters with genes not previously associated with virulence in the species. Examples of the latter were genes for pilus biosynthesis of the usher-chaperone (CU) pathway, for spermidine synthesis, and for effector proteins of the Type VI secretion system. Finally we leveraged on the clinically enriched genes to design PCR primers for detection and surveillance of pathogenic V. vulnificus strains.

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transi-tions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive chal-lenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome tran-sitions, resulting in species-level separation between aquatic biomes.