Background: Shallow coastal habitats are characterized by diverse macrophytes and often feature steep abiotic gradients, including salinity variations, which can shape the leaf- surface epi-microbiome (phyllosphere). To elucidate the effect of salinity and host identity on the phyllosphere of aquatic macrophytes in shallow water, we sampled the leaf surface microbiota across a salinity range of 6–15. Samples included the eelgrass Zostera marina, as well as the Eurasian water milfoil (Myriophyllum spicatum), muskgrass (Chara spp.), and sago pondweed (Stuckenia pectinata) in the brackish Baltic Sea during the summer of 2022. Microbial communities were characterized using 16S and 18S rRNA gene amplicon sequencing. Result: As hypothesized, the phyllosphere bacterial and protist community composition was distinct from the surrounding seawater microbiome. Typically associated taxa included the genera Loktanella, Pseudorhodobacter, the methylotrophic genus Methylotenera, unclassified Synechococcales, and Rhodobacteriaceae. Protist genera such as Picochlorum were consistently detected across all macrophyte hosts, while Cocconeis, Cyclotella, Mondous and unclassified Bacillariophyceae were present in all phyllospheres except Chara spp. Both, salinity and host species significantly influenced the composition and prevalence of the microbiota, primarily through shifts in the abundance of typical phyllosphere taxa. However, only 4–11% of phyllosphere taxa were uniquely associated with a specific salinity or macrophyte host. Conclusions: Our results demonstrate that aquatic macrophytes harbor a distinct and characteristic phyllosphere microbiome. The low proportion of host- or salinity specific taxa suggests that the most abundant members of this community are generalists, broadly adapted to the phyllosphere niche rather than being narrowly specialized. This implies that the presence of the macrophyte itself, providing a stable, nutrient-rich surface, exerts a stronger deterministic influence on the microbial community than the host identity or salinity fluctuations. Consequently, the phyllosphere appears relatively resilient to environmental variability, particularly salinity fluctuations. This highlights the robust nature of host-microbiome interactions and their importance for conservation of aquatic macrophyte ecosystems.

Microbial communities respond to physicochemical changes in the environment, making the microbiome a sensitive indicator of ecosystem status. Monitoring aquatic microbiomes is therefore essential for understanding ecosystem health and responses to change. While traditional monitoring relies on microscopy, DNA-based approaches that leverage advances in high-throughput sequencing are increasingly incorporated. Here, we evaluate the potential of using metabarcoding to predict abiotic and biotic parameters across spatiotemporal gradients of the Baltic Sea. The dataset comprises 397 seawater samples integrating prokaryotic and eukaryotic (16S and 18S rRNA gene) metabarcoding data with environmental measurements and plankton microscopy counts. Random Forest models based on metabarcoding data accurately predicted multiple physicochemical parameters and performed comparably to two other machine learning methods, XGBoost and TabPFN. Models using 16S rRNA gene data performed better than those using 18S rRNA gene data, with amplicon sequence variant-level yielding the most accurate results. Metabarcoding also exceeded plankton microscopy in predicting abiotic factors and effectively predicted the presence of phytoplankton and zooplankton genera from ≤1 L of water. Models trained on independent datasets accurately predicted several physicochemical parameters, though performance decreased for others highlighting challenges in transferability. Finally, metabarcoding-based predictions closely matched established eutrophication indicators of environmental status, demonstrating the utility of microbiome-based approaches for marine ecosystem monitoring and management.

Background: Periodontitis (PD) and rheumatoid arthritis (RA) are chronic inflammatory conditions, characterized by dysregulated immune response and excessive production of inflammatory mediators. The oral disease PD is triggered by periodontal pathogens, leading to the destruction of tissues surrounding the teeth, whereas RA is a systemic autoimmune disease primarily affecting the joints. The objective of this study was to investigate the prevalence of PD and map the profile of salivary and serum inflammatory mediators of patients with RA, with respect to periodontal severity (PD stage II and PD stage III/IV). Methods: For this cross-sectional cohort study, 62 patients diagnosed with RA were recruited. All participants underwent a full-mouth dental examination. Levels of various inflammatory mediators, including tumor necrosis factor (TNF) superfamily proteins, interferon (IFN) family proteins, regulatory T cell (Treg) cytokines, and matrix metalloproteinases were determined in saliva and serum samples from each participant using a human inflammation multiplex immunoassay panel. Results: In the current RA cohort, all participants were diagnosed with PD, of which 35.5% were classified as PD stage II and 64.5% as PD stages III/IV. Inflammatory mediator levels were significantly higher in both saliva and serum samples from patients with RA and PD stages III/IV, compared to those with RA and stage II within the same cohort. These included higher serum levels of sCD30, IL-10, IL-19, osteopontin and elevated salivary levels of BAFF/TNFSF13B and IFN-α2. Additionally, APRIL/TNFSF13 levels were increased in both saliva and serum. Conclusions: Among the studied patients with RA, the majority exhibited severe PD (stage III/IV), underscoring the importance of periodontal prophylaxis and treatment for this group of patients. Higher levels of inflammatory mediators were observed in both saliva and serum in those with PD stages III/IV, suggesting a potential link between the severity of PD and systemic inflammation in RA. Further research is needed to explore the clinical implications of these findings.

BackgroundZostera marina is an important ecosystem engineer influencing shallow water environments and possibly shaping the microbiota in surrounding sediments and water. Z. marina is typically found in marine systems, but it can also proliferate under brackish conditions. Changes in salinity generally have a strong impact on the biota, especially at the salty divide between salinity 6 and 9. To better understand the impact of the salty divide on the interaction between Z. marina and the surrounding sediment and water microbiota, we investigated the effects of Z. marina meadows on the surrounding microbiota across a salinity range of 6-15 in the Baltic Sea during the summer using 16S and 18S rRNA gene amplicon sequencing.ResultsSalinity was the most important factor for structuring the microbiota within both water and sediment. The presence of Z. marina affected the composition of the bacterial and eukaryotic community and bacterial alpha diversity in the sediment. However, this effect was confined to alpha-mesohaline conditions (salinity 9-15). The impact of Z. marina below salinity 9 on water and sediment microbiota was insignificant.ConclusionsIncreasing salinity was associated with a longer leaf length of Z. marina, causing an increased canopy height, which affects the sediment microbiota through reduced water velocity. Hence, we propose that the canopy effect may be the major predictor explaining Z. marina's interactions with the surrounding microbiota at salinity 9-15. These findings emphasize the importance of the physical effects of Z. marina meadow ecosystem services and have important implications for Z. marina management under brackish conditions in a changing climate.

Microorganisms thrive in complex communities shaped by intricate interactions, yet the extent and ecological implications of biosynthetic dependencies in natural communities remain underexplored. Here, we used a dilution approach to cultivate 204 microbial model communities from the Baltic Sea and recovered 527 metagenome-assembled genomes (MAGs) that dereplicated into 72 species-clusters (>95% average nucleotide identity, ANI). Of these species, at least 70% represent previously uncultivated lineages. Combined with 1073 MAGs from Baltic Sea metagenomes, we generated a genomic catalog of 701 species-clusters. Our results show that cultures with more than three species included microorganisms with smaller genome sizes, lower biosynthetic potential for amino acids and B vitamins, and higher prevalence and abundance in the environment. Moreover, the taxa found together in the same model communities had complementary biosynthetic gene repertoires. Our results demonstrate that cultivating bacteria in dilution model communities facilitates access to previously uncultivated but abundant species that likely depend on metabolic partners for survival. Together, our findings highlight the value of community-based cultivation for unraveling ecological strategies. Finally, we confirm that metabolic interdependencies and genome streamlining are widespread features of successful environmental microorganisms.

Marine ecosystems harbour a vast microbial diversity which play a crucial role in ecosystemfunctioning. Advancements in DNA sequencing technologies have transformed our ability to analyse microbial populations comprehensively. Metagenomic sequencing has emerged as a pivotal tool for characterising microbial communities across various environments. Bioinformatics, an interdisciplinary field, facilitates the analysis and interpretation of large biological datasets, including microbiome data.

This thesis aims to enhance bioinformatics approaches for analysing marine microbiomes. It comprises four papers covering bioinformatic developments and genomic data analysis across multiple topics, including metagenomics, pangenomics, comparative genomics and population genomics:

Paper I evaluated three assembly strategies for constructing gene catalogues from metagenomic samples: individual sample assembly with gene clustering, co-assembly of all samples, and a new hybrid approach, mix assembly. The efficacy of the mix-assembly approach was highlighted for maximising information extraction from metagenomic samples, offering opportunities for further exploration in microbial ecology and environmental genomics.

Using the mix-assembly approach, we conducted a comprehensive analysis of 124 metagenomic samples sourced from the Baltic Sea, resulting in the refinement of the Baltic Sea Gene Set (BAGS v1.1), which now encompasses 66.53 million genes annotated for both functionality and taxonomy. In Paper II, we introduced an open-access initiative that provided the mix-assembly pipeline code. We also developed the BAGS-Shiny web application to facilitate user interaction with this extensive gene catalogue.

Paper III focused on whole-genome sequencing and assembly of 82 environmental V. vulnificus strains from the Baltic Sea, enabling comprehensive comparative genomic analysis. I developed the PhyloBOTL pipeline, which uses a phylogeny-based approach to identify genes associated with pathogenicity. Comparative genomics of 208 clinical isolates and 199 environmental isolates revealed 58 enriched orthologs in pathogenic strains, including known virulence factors and novel genes. Potential biomarkers for pathogenic V. vulnificus were identified, and primers suitable for PCR-based environmental monitoring were designed (in silico).

In Paper IV population genomics analysis was carried out, using the Input_Pogenom pipeline and POGENOM tool, to explore intraspecific biogeographical patterns. Geographical barriers were found to significantly influence aquatic bacteria distribution, with greater genetic differentiation observed between Baltic and Caspian seas than within the Baltic Sea's salinity gradient.

Havsmiljöer hyser en enorm mikrobiell mångfald som spelar en avgörande roll för ekosystemens funktion. Framsteg inom DNA-sekvenseringstekniker har revolutionerat vår förmåga att analysera

den mikrobiella populationen på ett omfattande sätt. Metagenomisk sekvensering har framträtt som ett centralt verktyg för att karakterisera mikrobiella samhällen i olika miljöer. Bioinformatik, ett tvärvetenskapligt fält, underlättar analys och tolkning av stora biologiska dataset, inklusive mikrobiomdata.

Den här avhandlingen syftar till att förbättra bioinformatiska metoder för att analysera marina mikrobiom. Den består av fyra artiklar som täcker bioinformatisk utveckling och analys av genomdata inom flera områden, inklusive metagenomik, pangenom, jämförande genomik och populationsgenetik:

Artikel I utvärderade tre monteringsstrategier för att konstruera genkataloger från metagenomiska prover: montering av enskilda prover med genglustering, sammontering av alla prover och en ny hybridmetod, mixmontering. Effektiviteten hos mixmonteringsmetoden lyftes fram för att maximera informationsutvinning från metagenomiska prover, vilket öppnar för vidare utforskning inom mikrobiell ekologi och miljögenomik.

Med hjälp av mixmonteringsmetoden genomförde vi en omfattande analys av 124 metagenomiska prover från Östersjön, vilket resulterade i förfiningen av Östersjöns gensets (BAGS v1.1), som nu omfattar 66,53 miljoner gener annoterade för både funktion och taxonomi. I artikel II introducerade vi ett öppet initiativ som tillhandahöll koden för mixmonterings-pipeline. Vi utvecklade också BAGS-Shiny webbapplikationen för att underlätta användarinteraktion med denna omfattande gensetskatalog.

Artikel III fokuserade på helgenomsekvensering och montering av 82 miljörelaterade V. vulnificus-stammar från Östersjön, vilket möjliggjorde omfattande jämförande genomisk analys.

Jag utvecklade PhyloBOTL-pipelinen, som använder en fylogenibaserad metod för att identifiera gener associerade med patogenicitet. Jämförande genomik av 208 kliniska isolat och 199 miljöisolerade isolat avslöjade 58 anrikade ortologer i patogena stammar, inklusive kända virulensfaktorer och nya gener. Potentiella biomarkörer för patogena V. vulnificus identifierades och primers lämpliga för PCR-baserad miljöövervakning designades (in silico).

I artikel IV utfördes populationsgenetisk analys med hjälp av Input_Pogenom-pipelinen och POGENOM-verktyget för att utforska intraspecifika biogeografiska mönster. Geografiska barriärer visade sig signifikant påverka vattenlevande bakteriers utbredning, med större genetisk differentiering observerad mellan Östersjön och Kaspiska havet än inom Östersjöns salthaltsgradient.

Due to climate change the pathogenic bacterium Vibrio vulnificus proliferates along brackish coastlines, posing risks to public health, tourism, and aquaculture. Here we investigated previously suggested regulation measures to reduce the prevalence of V. vulnificus, locally through seagrass and regionally through the reduction of eutrophication and consequential formation of algal blooms. Field samples collected in the summer of 2021 covered the salinity and eutrophication gradients of the Baltic Sea, one of the largest brackish areas worldwide. Physico-, biological- and hydrochemical parameters were measured and variables explaining V. vulnificus occurrence were identified by machine learning. The best V. vulnificus predictors were eutrophication-related features, such as particulate organic carbon and nitrogen, as well as occurrence of potential phytoplankton blooms and associated species. V. vulnificus abundance did not vary significantly between vegetated and non-vegetated areas. Thus, reducing nutrient inputs could be an effective method to control V. vulnificus populations in eutrophied brackish coasts.

The number of Vibrio-related infections in humans, e.g., by Vibrio vulnificus, has increased along the coasts of the Baltic Sea. Due to climate change, vibriosis risk is expected to increase. It is, therefore, pertinent to design a strategy for mitigation of the vibriosis threat in the Baltic Sea area, but a prerequisite is to identify the environmental conditions promoting the occurrence of pathogenic Vibrio spp., like V. vulnificus. To address this, we sampled three coastal Baltic sites in Finland, Germany, and Denmark with salinities between 6 and 21 from May to October 2022. The absolute and relative abundances of Vibrio spp. and V. vulnificus in water were compared to environmental conditions, including the presence of the eelgrass Zostera marina, which has been suggested to reduce pathogenic Vibrio species abundance. In the water column, V. vulnificus only occurred at the German station between July and August at salinity 8.1-11.2. Temperature and phosphate (PO43-) were identified as the most influencing factors for Vibrio spp. and V. vulnificus. The accumulation of Vibrio spp. in the sediment and the co-occurrence with sediment bacteria in the water column indicate that sediment resuspension contributed to V. vulnificus abundance. Interestingly, V. vulnificus co-occurred with specific cyanobacteria taxa, as well as specific bacteria associated with cyanobacteria. Although we found no reduction in Vibrio spp. or V. vulnificus associated with eelgrass beds, our study underscores the importance of extended heatwaves and sediment resuspension, which may elevate the availability of PO43-, for Vibrio species levels at intermediate salinities in the Baltic Sea. IMPORTANCE: Elevated sea surface temperatures are increasing the prevalence of pathogenic Vibrio at higher latitudes. The recent increase in Vibrio-related wound infections and deaths along the Baltic coasts is, therefore, of serious health concern. We used culture-independent data generated from three Baltic coastal sites in Denmark, Germany, and Finland from May to October (2022), with a special focus on Vibrio vulnificus, and combined it with environmental data. Our temporal model shows that temperature, combined with sediment resuspension, drives the prevalence of V. vulnificus at intermediate salinities in the coastal Baltic Sea.

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.

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.