Microbial communities are the main drivers of biogeochemical cycling of multiple elements sustaining life in the ocean. The rapidity of their response to stressors and abrupt environmental changes implies that even fast and infrequent events can affect local transformations of organic matter and nutrients. Modern molecular techniques now allow for monitoring of microbial activities and functions in the environment through the analysis of genes and expressed genes contained in natural microbial assemblages. However, messenger RNA turnover in cells can be as short as 30 seconds and stability varies greatly between transcripts. Sampling of in situ communities involves an inevitable delay between the collection of seawater and the extraction of its RNA, leaving the bacterial communities plenty of time to alter their gene expression. The characteristics of microbial RNA turnover make time-series very difficult because samples need to be processed immediately to limit alterations to the metatranscriptomes. To address these challenges we designed an autonomous in situ fixation multi-sampler (AFISsys) for the reliable sampling of microbial metatranscriptomes at frequent intervals, for refined temporal resolution. To advance the development of this instrument, we examined the minimal seawater volume necessary for adequate coverage of community gene expression, and the suitability of phenol/ethanol fixation for immediate and long-term preservation of transcripts from a microbial community. We then evaluated the field eligibility of the instrument itself, with two case studies in a brackish system. AFISsys is able to collect, fix, and store water samples independently at a predefined temporal resolution. Phenol/ethanol fixation can conserve metatranscriptomes directly in the environment for up to a week, for later analysis in the laboratory. Thus, the AFISsys constitutes an invaluable tool for the integration of molecular functional analyses in environmental monitoring in brackish waters and in aquatic environments in general. 

Atmospheric deposition is a source of inorganic nutrients and organic matter to the ocean, and can favor the growth of some planktonic species over others according to their nutrient requirements. Atmospheric inputs from natural and anthropogenic sources are nowadays increasing due to desertification and industrialization, respectively. While the impact of mineral dust (mainly from the Saharan desert) on phytoplankton and bacterial community composition has been previously assessed, the effect of anthropogenic aerosols on marine bacterial assemblages remains poorly studied. Since marine bacteria play a range of roles in the biogeochemical cycles of inorganic nutrients and organic carbon, it is important to determine which taxa of marine bacteria may benefit from aerosol fertilization and which not. Here, we experimentally assessed the effect of Saharan dust and anthropogenic aerosols on marine bacterioplankton community composition across a spatial and temporal range of trophic conditions in the northwestern Mediterranean Sea. Results from 16S rDNA sequencing showed that bacterial diversity varied significantly with seasonality and geographical location. While atmospheric deposition did not yield significant changes in community composition when all the experiments where considered together, it did produce changes at certain places and during certain times of the year. These effects accounted for shifts in the bacterial community's relative abundance of up to 28%. The effect of aerosols was overall greatest in summer, both types of atmospheric particles stimulating the groups Alphaproteobacteria, Betaproteobacteria, and Cyanobacteria in the location with the highest anthropogenic footprint. Other bacterial groups benefited from one or the other aerosol depending on the season and location. Anthropogenic aerosols increased the relative abundance of groups belonging to the phylum Bacteriodetes (Cytophagia, Flavobacteriia, and Sphingobacteriia), while Saharan dust stimulated most the phytoplanktonic group of Cyanobacteria and, more specifically, Synechococcus.

Heterotrophic bacteria are important drivers of nitrogen (N) cycling and the processing of dissolved organic matter (DOM). Projected increases in precipitation will potentially cause increased loads of riverine DOM to the Baltic Sea and likely affect the composition and function of bacterioplankton communities. To investigate this, the effects of riverine DOM from two different catchment areas (agricultural and forest) on natural bacterioplankton assemblages from two contrasting sites in the Baltic Sea were examined. Two microcosm experiments were carried out, where the community composition (16S rRNA gene sequencing), the composition of a suite of N-cycling genes (metagenomics) and the abundance and transcription of ammonia monooxygenase (amoA) genes involved in nitrification (quantitative PCR) were investigated. The river water treatments evoked a significant response in bacterial growth, but the effects on overall community composition and the representation of N-cycling genes were limited. Instead, treatment effects were reflected in the prevalence of specific taxonomic families, specific N-related functions and in the transcription of amoA genes. The study suggests that bacterioplankton responses to changes in the DOM pool are constrained to part of the bacterial community, whereas most taxa remain relatively unaffected.

Periodontitis is a microbial-induced chronic inflammatory disease, which may not only result in tooth loss, but can also contribute to the development of various systemic diseases. The transition from healthy to diseased periodontium depends on microbial dysbiosis and impaired host immune response. Although periodontitis is a common disease as well as associated with various systemic inflammatory conditions, the taxonomic profiling of the salivary microbiota in periodontitis and its association with host immune and inflammatory mediators has not been reported. Therefore, the aim of this study was to identify key pathogens and their potential interaction with the host's inflammatory mediators in saliva samples for periodontitis risk assessment. The microbial 16S rRNA gene sequencing and the levels of inflammatory mediators were performed in saliva samples from patients with chronic periodontitis and periodontally healthy control subjects. The salivary microbial community composition differed significantly between patients with chronic periodontitis and healthy controls. Our analyses identified a number of microbes, including bacteria assigned to Eubacterium saphenum, Tannerella forsythia, Filifactor alocis, Streptococcus mitis/parasanguinis, Parvimonas micra, Prevotella sp., Phocaeicola sp., and Fretibacterium sp. as more abundant in periodontitis, compared to healthy controls. In samples from healthy individuals, we identified Campylobacter concisus, and Veillonella sp. as more abundant. Integrative analysis of the microbiota and inflammatory mediators/cytokines revealed associations that included positive correlations between the pathogens Treponema sp. and Selenomas sp. and the cytokines chitinase 3-like 1, sIL-6R alpha, sTNF-R1, and gp 130/sIL-6R beta. In addition, a negative correlation was identified between IL-10 and Filifactor alocis. Our results reveal distinct and disease-specific patterns of salivary microbial composition between patients with periodontitis and healthy controls, as well as significant correlations between microbiota and host-mediated inflammatory cytokines. The positive correlations between the pathogens Treponema sp. and Selenomas sp. and the cytokines chitinase 3-like 1, sIL-6R alpha, sTNF-R1, and gp 130/sIL-6R beta might have the future potential to serve as a combined bacteria-host salivary biomarker panel for diagnosis of the chronic infectious disease periodontitis. However, further studies are required to determine the capacity of these microbes and inflammatory mediators as a salivary biomarker panel for periodontitis.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter. In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures. IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle effect") may occur due to the enclosure itself. In this study, we analyzed the bacterial communities that originated from three different salinities of the Baltic Sea, comparing their compositions and physiological activities both at the early stage and after 55 days of incubation. Our results suggested that internal carbon storage strategies impact the success of certain bacterial species, independent of the experimental manipulation. Thus, while enclosure experiments remain valid tools in environmental research, microbial community composition shifts must be critically followed. This investigation of the metaproteome during long-term batch enclosures expanded our current understanding of the so-called "bottle effect," which is well known to occur during enclosure experiments.

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the "barcoding region" of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein-coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon-based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.

The Baltic Sea is one of the world's largest brackish water bodies and is characterised by pronounced physicochemical gradients where microbes are the main biogeochemical catalysts. Meta-omic methods provide rich information on the composition of, and activities within, microbial ecosystems, but are computationally heavy to perform. We here present the Baltic Sea Reference Metagenome (BARM), complete with annotated genes to facilitate further studies with much less computational effort. The assembly is constructed using 2.6 billion metagenomic reads from 81 water samples, spanning both spatial and temporal dimensions, and contains 6.8 million genes that have been annotated for function and taxonomy. The assembly is useful as a reference, facilitating taxonomic and functional annotation of additional samples by simply mapping their reads against the assembly. This capability is demonstrated by the successful mapping and annotation of 24 external samples. In addition, we present a public web interface, BalticMicrobeDB, for interactive exploratory analysis of the dataset.

Olkiluoto, an island on the south-west coast of Finland, will host a deep geological repository for the storage of spent nuclear fuel. Microbially induced corrosion from the generation of sulphide is therefore a concern as it could potentially compromise the longevity of the copper waste canisters. Groundwater at Olkiluoto is geochemically stratified with depth and elevated concentrations of sulphide are observed when sulphate-rich and methane-rich groundwaters mix. Particularly high sulphide is observed in methane-rich groundwater from a fracture at 530.6 mbsl, where mixing with sulphate-rich groundwater occurred as the result of an open drill hole connecting two different fractures at different depths. To determine the electron donors fuelling sulphidogenesis, we combined geochemical, isotopic, metagenomic and metaproteomic analyses. This revealed a low diversity microbial community fuelled by hydrogen and organic carbon. Sulphur and carbon isotopes of sulphate and dissolved inorganic carbon, respectively, confirmed that sulphate reduction was ongoing and that CO2 came from the degradation of organic matter. The results demonstrate the impact of introducing sulphate to a methane-rich groundwater with limited electron acceptors and provide insight into extant metabolisms in the terrestrial subsurface. 

The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming.

Bacteria are major drivers of biogeochemical nutrient cycles and energy fluxes in marine environments, yet how bacterial communities respond to environmental change is not well known. Metagenomes allow examination of genetic responses of the entire microbial community to environmental change. However, it is challenging to link metagenomes directly to biogeochemical process rates. Here, we investigate metagenomic responses in natural bacterioplankton communities to simulated environmental stressors in the Baltic Sea, including increased river water input, increased nutrient concentration, and reduced oxygen level. This allowed us to identify informative prokaryotic gene markers, responding to environmental perturbation. Our results demonstrate that metagenomic and metabolic changes in bacterial communities in response to environmental stressors are influenced both by the initial community composition and by the biogeochemical factors shaping the functional response. Furthermore, the different sources of dissolved organic matter (DOM) had the largest impact on metagenomic blueprint. Most prominently, changes in DOM loads influenced specific transporter types reflecting the substrate availability and DOC assimilation and consumption pathways. The results provide new knowledge for developing models of ecosystem structure and biogeochemical cycling in future climate change scenarios and advance our exploration of the potential use of marine microorganisms as markers for environmental conditions.