Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea.

Vibrio species are ubiquitously distributed in marine waters all over the world. High genome plasticity due to frequent mutation, recombination, and lateral gene transfer enables Vibrio to adapt rapidly to environmental changes. The genus Vibrio comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions. In recent years, pathogenic Vibrio emerged also in coastal European waters. Little is known about factors driving the proliferation of Vibrio spp. in temperate waters such as the North Sea. In this study a quantification of Vibrio in the North Sea and their response to biotic and abiotic parameters were assessed. Between January and December 2009, Vibrio at Helgoland Roads (North Sea, Germany) were quantified using fluorescence in situ hybridization. Vibrio numbers up to 3.4 × 10(4) cells × mL(-1) (2.2% of total microbial counts) were determined in summer, but their abundance was significantly lower in winter (5 × 10(2) cells × mL(-1)). Correlations between Vibrio and nutrients (SiO(2), PO(4) (3-), DIN), Secchi depth, temperature, salinity, and chlorophyll a were calculated using Spearman rank analysis. Multiple stepwise regression analysis was carried out to analyze the additive influence of multiple factors on Vibrio. Based on these calculations, we found that high water temperature and low salinity best explained the increase of Vibrio cell numbers. Other environmental parameters, especially nutrients and chlorophyll a, also had an influence. All variables were shown to be subject to the overall seasonal dynamics at Helgoland Roads. Multiple regression models could represent an efficient and reliable tool to predict Vibrio abundances in response to the climate change in European waters.

The travelling particles: community dynamics of biofilms on microplastics transferred along a salinity gradient.

Microplastics (MP), as novel substrata for microbial colonization within aquatic ecosystems, are a matter of growing concern due to their potential to propagate foreign or invasive species across different environments. MP are known to harbour a diversity of microorganisms, yet little is understood of the dynamics of their biofilms and their capacity to successfully displace these microorganisms across different aquatic ecosystems typically marked by steep salinity gradients. To address this, we performed an in situ sequential incubation experiment to simulate MP transport from riverine to coastal seawaters using synthetic (high-density polyethylene, HDPE and tyre wear, TW) and natural (Wood) substrata. Bacterial communities on incubated particles were compared to each other as well as to those in surrounding waters, and their dynamics along the gradient investigated. All communities differed significantly from each other in their overall structure along the salinity gradient and were shaped by different ecological processes. While HDPE communities were governed by environmental selection, those on TW and Wood were dominated by stochastic events of dispersal and drift. Upon transfer into coastal seawaters, an almost complete turnover was observed among HDPE and TW communities. While synthetic particles displaced a minor proportion of communities across the salinity gradient, some of these comprised putatively pathogenic and resistant taxa. Our findings present an extensive assessment of MP biofilms and their dynamics upon displacement across different aquatic systems, presenting new insights into the role of MP as transport vectors.

Shell Disease Syndrome Is Associated with Reduced and Shifted Epibacterial Diversity on the Carapace of the Crustacean Cancer pagurus.

Cancer pagurus is highly susceptible to shell disease syndrome. However, little is known about concomitant changes in the epibacterial community. We compared the bacterial communities of black spot affected and nonaffected areas of the carapace by amplicon sequencing of 16S rRNA genes and 16S rRNA. Within each spot, bacterial communities of affected areas were less diverse compared to communities from nonaffected areas. Communities of different affected spots were, however, more divergent from each other, compared to those of different nonaffected areas. This indicates a reduced and shifted microbial community composition caused by the black spot disease. Different communities found in black spots likely indicate different stages of the disease. In affected areas, Flavobacteriaceae rose to one of the most abundant and active families due to the increase of Aquimarina spp., suggesting a significant role in shell disease syndrome. We isolated 75 bacterial strains from diseased and healthy areas, which are primarily affiliated with Proteobacteria and Bacteroidetes, reflecting the dominant phyla detected by amplicon sequencing. The ability to degrade chitin was mainly found for Gammaproteobacteria and Aquimarina spp. within the Flavobacteriia, while the ability to use N-acetylglucosamine, the monomer of the polysaccharide chitin, was observed for most isolates, including many Alphaproteobacteria. One-third of the isolates, including most Aquimarina spp., showed antagonistic properties, indicating a high potential for interactions between the bacterial populations. The combination of bacterial community analysis and the physiological properties of the isolates provided insights into a functional complex epibacterial community on the carapace of C. pagurus. IMPORTANCE In recent years, shell disease syndrome has been detected for several ecologically and economically important crustacean species. Large proportions of populations are affected, e.g., >60% of the widely distributed species Cancer pagurus in different North Sea areas. Bacteria play a significant role in the development of different forms of shell disease, all characterized by microbial chitinolytic degradation of the outer shell. By comparing the bacterial communities of healthy and diseased areas of the shell of C. pagurus, we demonstrated that the disease causes a reduced bacterial diversity within affected areas, a phenomenon co-occurring also with many other diseases. Furthermore, the community composition dramatically changed with some taxa rising to high relative abundances and showing increased activity, indicating strong participation in shell disease. Characterization of bacterial isolates obtained from affected and nonaffected spots provided deeper insights into their physiological properties and thus the possible role within the microbiome.

Highly diverse flavobacterial phages isolated from North Sea spring blooms.

It is generally recognized that phages are a mortality factor for their bacterial hosts. This could be particularly true in spring phytoplankton blooms, which are known to be closely followed by a highly specialized bacterial community. We hypothesized that phages modulate these dense heterotrophic bacteria successions following phytoplankton blooms. In this study, we focused on Flavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms. Taxonomic analysis allowed us to delineate ten new phage genera and ten new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating showed that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years suggested that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.

Temporal variability of coastal Planctomycetes clades at Kabeltonne station, North Sea.

Members of the bacterial phylum Planctomycetes are reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution of Planctomycetes in surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Generally Planctomycetes are more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed that Planctomycetes abundance was correlated to the Centrales diatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ~90% of the Planctomycetes reside in the >3-µm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, named Planctomyces-related group A, uncultured Planctomycetes group B, and Pirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. Uncultured Planctomycetes group B was most abundant in autumn samples, while Planctomyces-related group A was present in high numbers only during late autumn and winter. The levels of Pirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of the Planctomycetes is correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle.

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10(3) N l(-1) and MPNs up to 240 N g(-1) were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.

Mycoplankton Biome Structure and Assemblage Processes Differ Along a Transect From the Elbe River Down to the River Plume and the Adjacent Marine Waters.

Rivers are transport systems and supply adjacent ecosystems with nutrients. They also serve human well-being, for example as a source of food. Microorganism biodiversity is an important parameter for the ecological balance of river ecosystems. Despite the knowledge that fungi are key players in freshwater nutrient cycling and food webs, data on planktonic fungi of streams with higher stream order are scarce. This study aims to fill this knowledge gap by a fungi-specific 18S ribosomal RNA (rRNA) gene tag sequencing approach, investigating mycoplankton diversity in the Elbe River along a transect from shallow freshwater, to the estuary and river plume down to the adjacent marine waters (sections of seventh stream order number). Using multivariate analyses and the quantitative process estimates (QPEs) method, questions (i) of how mycoplankton communities as part of the river continuum change along the transect, (ii) what factors, spatial and environmental, play a role, and (iii) what assembly processes, such as selection or dispersion, operate along the transect, were addressed. The partitioning of mycoplankton communities into three significant distant biomes was mainly driven by local environmental conditions that were partly under spatial control. The assembly processes underlying the biomes also differed significantly. Thus, variable selection dominated the upstream sections, while undominated processes like ecological drift dominated the sections close to the river mouth and beyond. Dispersal played a minor role. The results suggest that the ecological versatility of the mycoplankton communities changes along the transect as response, for example, to a drastic change from an autotrophic to a heterotrophic system caused by an abrupt increase in the river depth. Furthermore, a significant salinity-dependent occurrence of diverse basal fungal groups was observed, with no clade found exclusively in marine waters. These results provide an important framework to help understand patterns of riverine mycoplankton communities and serve as basis for a further in-depth work so that fungi, as an important ecological organism group, can be integrated into models of, e.g., usage-balance considerations of rivers.

Schoolchildren discover hotspots of floating plastic litter in rivers using a large-scale collaborative approach.

Rivers are an important transport route of anthropogenic litter from inland sources toward the sea. A collaborative (i.e. citizen science) approach was used to evaluate the litter pollution of rivers in Germany: schoolchildren within the project "Plastic Pirates" investigated rivers across the entire country during the years 2016 and 2017 by surveying floating macrolitter at 282 sites and taking 164 meso-/microplastic samples (i.e. particles 24.99-5 mm, and 4.99-1 mm, respectively). Floating macrolitter was sighted at 54% of sampling sites and floating macrolitter quantities ranged from 0 to 8.25 items m-1 h-1 (average of 0.34 ± 0.89 litter items m-1 h-1). Floating meso-/microplastics were present at 57% of the sampling sites, and floating meso-/microplastic quantities ranged from 0 to 220 particles h-1 (average of 6.86 ± 24.11 items h-1). As only particles >1 mm were sampled and analyzed, the pollution of rivers in Germany by microplastics could be a much more prevalent problem, regardless of the size of the river. We identified six plastic pollution hotspots where 60% of all meso-/microplastics collected in the present study were found. These hotspots were located close to a plastic-producing industry site, a wastewater treatment plant, at and below weirs, or in residential areas. The composition of the particles at these hotspots indicates plastic producers and possibly the construction industry and wastewater treatment plants as point sources. An identification of litter hotspots would enable specific mitigation measures, adjusted to the respective source, and thereby could prevent the release of large quantities of small plastic particles in rivers. The adopted large-scale citizen science approach was especially suitable to detect pollution hotspots by sampling a variety of rivers, large and small, and enabled a national overview of litter pollution in German rivers.

Diatom fucan polysaccharide precipitates carbon during algal blooms.

The formation of sinking particles in the ocean, which promote carbon sequestration into deeper water and sediments, involves algal polysaccharides acting as an adhesive, binding together molecules, cells and minerals. These as yet unidentified adhesive polysaccharides must resist degradation by bacterial enzymes or else they dissolve and particles disassemble before exporting carbon. Here, using monoclonal antibodies as analytical tools, we trace the abundance of 27 polysaccharide epitopes in dissolved and particulate organic matter during a series of diatom blooms in the North Sea, and discover a fucose-containing sulphated polysaccharide (FCSP) that resists enzymatic degradation, accumulates and aggregates. Previously only known as a macroalgal polysaccharide, we find FCSP to be secreted by several globally abundant diatom species including the genera Chaetoceros and Thalassiosira. These findings provide evidence for a novel polysaccharide candidate to contribute to carbon sequestration in the ocean.

Constitutive expression of the proteorhodopsin gene by a flavobacterium strain representative of the proteorhodopsin-producing microbial community in the North Sea.

Proteorhodopsin (PR), a photoactive proton pump containing retinal, is present in approximately half of all bacteria in the ocean, but its physiological role is still unclear, since very few strains carrying the PR gene have been cultured. The aim of this work was to characterize PR diversity in a North Sea water sample, cultivate a strain representative of North Sea PR clusters, and study the effects of light and carbon concentration on the expression of the PR gene. A total of 117 PR sequences, of which 101 were unique, were obtained from a clone library of PCR-amplified PR gene fragments. Of the North Sea PRs, 97% were green light absorbing, as inferred from the amino acid at position 105; 67% of the PR protein fragments showed closest similarity to PRs from Alphaproteobacteria, 4% showed closest similarity to PRs from Gammaproteobacteria, and 29% showed closest similarity to PRs from "Bacteroidetes"/Flavobacteria. The dominant PR cluster (comprising 18% of all PRs) showed a high degree of similarity to the PR from the cultivated Roseobacter strain HTCC2255. The relative abundances of the North Sea PR clusters were confirmed by quantitative PCR. They were detected in metagenomic fragments from coastal oceans worldwide with various degrees of abundance. Several hundred bacterial strains from the North Sea water sample were cultivated on oligocarbophilic media. By screening with degenerate primers, two strains carrying the PR gene were identified. Their 16S rRNA gene sequences were identical and affiliated with a Bacteroidetes subcluster from the North Sea. The PR sequence of isolate PRO95 was completed by chromosomal walking. It was 76% identical to that of Dokdonia donghaensis MED134 and was functional, as indicated by the signature amino acids. PRO95 expressed its PR gene in liquid media containing between 9.7 and 121 mM carbon, both in the light and in the dark. Growth was not enhanced by light. Thus, the detection of the physiological role of PR may require more sensitive methods.

Looking for the hidden: Characterization of lysogenic phages in potential pathogenic Vibrio species from the North Sea.

The incidence of potentially pathogenic Vibrio species in the marine environment around Europe, is correlated with the increase of surface seawater temperature. Despite their importance, little is known about the trigger factors of potential outbreak-causing strains in this region. As prophages may compose a major reservoir of virulence traits in marine ecosystems, this study aims to identify and characterize the genomes of lysogenic Vibrio phages exemplarily from the North Sea. Therefore, 31 isolates from potentially pathogenic Vibrio species from the North Sea were screened for inducible prophages with mitomycin C. From them, one V. cholerae isolate and 40% V. parahaemolyticus isolates carried inducible prophages. Three lysogenic phages were selected for genomic characterization. The phage vB_VpaM_VP-3212 (unclassified Myoviridae) has a genome with a length of 36.81 Kbp and 55 CDS were identified. This lysogenic phage of V. parahaemolyticus contains genes related to replicative transposition mechanism, such as transposase and mobile elements similar to Mu-like viruses. The phage vB_VpaP_VP-3220 (Podoviridae, unclassified Nona33virus) has a genome length of 58,14 Kbp and contains 63 CDS. This V. parahaemolyticus phage probably uses a headful (pac) packaging replication mechanism. The phage vB_VchM_VP-3213 (unclassified Myoviridae) has a genome with a length of 41 Kbp and 63 CDS were identified, including integrase and Xer system for lysogenic recombination. This lysogenic phage of V. cholerae has similar genomic features as lambdoid phages. Although no pathogenicity genes were identified, their similarity among other phage genomes indicates that these phages can affect the development of pathogenic Vibrio strains in marine environments.

Genomic characterization of filamentous phage vB_VpaI_VP-3218, an inducible prophage of Vibrio parahaemolyticus.

The seawater temperature rise can promote the growth of potentially pathogenic Vibrio species. In the North Sea, V. parahaemolyticus strains have been isolated and characterized. These strains contain prophages that may contribute to the emergence of pathogenic strains in the marine environment. Here, we present the genome structure and possible biological functions of the inducible phage vB_VpaI_VP-3218, a novel filamentous phage carried by the V. parahaemolyticus strain VN-3218. Prophages of the strain VN-3218 were induced with mitomycin C and the DNA from the phage induction was sequenced. Two incomplete prophages were identified, only one complete phage genome with length of 11,082 bp was characterized. The phage vB_VpaI_VP-3218 belongs to the Inoviridae family and shows close homology to the Saetivirus genus. This phage can integrate into the chromosomal host genome and carries host-related regions absent in similar phage genomes, suggesting that this phage might integrate in other Vibrio host genomes from the environment. Furthermore, this phage might have a role in pathogenicity due to potential zonula occludens toxin genes. Based on its genomic similarity, the genome of vB_VpaI_VP-3218 phage probably integrates into the lysogen's chromosome and replicates as episome. This study complements prophage induction and bioinformatic studies applied to non-model species of potentially pathogenic Vibrio species. The characterization of this phage provides new insights with respect to the presence of filamentous phages in environmental V. parahaemolyticus strains, which might have a role in the emergence of new pathogenic strains in the North Sea.

Potentially human pathogenic Vibrio spp. in a coastal transect: Occurrence and multiple virulence factors.

An increase in human Vibrio spp. infections has been linked to climate change related events, in particular to seawater warming and heatwaves. However, there is a distinct lack of research of pathogenic Vibrio spp. occurrences in the temperate North Sea, one of the fastest warming seas globally. Particularly in the German Bight, Vibrio investigations are still scarce. This study focuses on the spatio-temporal quantification and pathogenic characterization of V. parahaemolyticus, V. vulnificus and V. cholerae over the course of 14 months. Species-specific MPN-PCR (Most probable number - polymerase chain reaction) conducted on selectively enriched surface water samples revealed seasonal patterns of all three species with increased abundances during summer months. The extended period of warm seawater coincided with prolonged Vibrio spp. occurrences in the German Bight. Temperature and nitrite were the factors explaining variations in Vibrio spp. abundances after generalized additive mixed models. The specific detection of pathogenic markers via PCR revealed trh-positive V. parahaemolyticus, pathogenic V. vulnificus (nanA, manIIA, PRXII) and V. cholerae serotype O139 presence. Additionally, spatio-temporally varying virulence profiles of V. cholerae with multiple accessory virulence-associated genes, such as the El Tor variant hemolysin (hlyAET), acyltransferase of the repeats-in-toxin cluster (rtxC), Vibrio 7th pandemic island II (VSP-II), Type III Secretion System (TTSS) and the Cholix Toxin (chxA) were detected. Overall, this study highlights that environmental human pathogenic Vibrio spp. comprise a reservoir of virulence-associated genes in the German Bight, especially in estuarine regions. Due to their known vast genetic plasticity, we point to the possible emergence of highly pathogenic V. cholerae strains. Particularly, the presence of V. cholerae serotype O139 is unusual and needs urgent continuous surveillance. Given the predictions of further warming and more frequent heatwave events, human pathogenic Vibrio spp. should be seriously considered as a developing risk to human health in the German Bight.

The Travelling Particles: Investigating microplastics as possible transport vectors for multidrug resistant E. coli in the Weser estuary (Germany).

The prevalence of multidrug-resistant Gram-negative bacteria in aquatic environments has been a long withstanding health concern, namely extended-spectrum beta-lactamase (ESBL) producing Escherichia coli. Given increasing reports on microplastic (MP) pollution in these environments, it has become crucial to better understand the role of MP particles as transport vectors for such multidrug-resistant bacteria. In this study, an incubation experiment was designed where particles of both synthetic and natural material (HDPE, tyre wear, and wood) were sequentially incubated at multiple sites along a salinity gradient from the Lower Weser estuary (Germany) to the offshore island Helgoland (German Bight, North Sea). Following each incubation period, particle biofilms and water samples were assessed for ESBL-producing E. coli, first by the enrichment and detection of E. coli using Fluorocult® LMX Broth followed by cultivation on CHROMAgar™ ESBL media to select for ESBL-producers. Results showed that general E. coli populations were present on the surfaces of wood particles across all sites but none were found to produce ESBLs. Additionally, neither HDPE nor tyre wear particles were found to harbour any E. coli. Conversely, ESBL-producing E. coli were present in surrounding waters from all sites, 64% of which conferred resistances against up to 3 other antibiotic groups, additional to the beta-lactam resistances intrinsic to ESBL-producers. This study provides a first look into the potential of MP to harbour and transport multidrug-resistant E. coli across different environments and the approach serves as an important precursor to further studies on other potentially harmful MP-colonizing species.

Seasonal Dynamics of Pelagic Mycoplanktonic Communities: Interplay of Taxon Abundance, Temporal Occurrence, and Biotic Interactions.

Marine fungi are an important component of pelagic planktonic communities. However, it is not yet clear how individual fungal taxa are integrated in marine processes of the microbial loop and food webs. Most likely, biotic interactions play a major role in shaping the fungal community structure. Thus, the aim of our work was to identify possible biotic interactions of mycoplankton with phytoplankton and zooplankton groups and among fungi, and to investigate whether there is coherence between interactions and the dynamics, abundance and temporal occurrence of individual fungal OTUs. Marine surface water was sampled weekly over the course of 1 year, in the vicinity of the island of Helgoland in the German Bight (North Sea). The mycoplankton community was analyzed using 18S rRNA gene tag-sequencing and the identified dynamics were correlated to environmental data including phytoplankton, zooplankton, and abiotic factors. Finally, co-occurrence patterns of fungal taxa were detected with network analyses based on weighted topological overlaps (wTO). Of all abundant and persistent OTUs, 77% showed no biotic relations suggesting a saprotrophic lifestyle. Of all other fungal OTUs, nearly the half (44%) had at least one significant negative relationship, especially with zooplankton and other fungi, or to a lesser extent with phytoplankton. These findings suggest that mycoplankton OTUs are embedded into marine food web chains via highly complex and manifold relationships such as parasitism, predation, grazing, or allelopathy. Furthermore, about one third of all rare OTUs were part of a dense fungal co-occurrence network probably stabilizing the fungal community against environmental changes and acting as functional guilds or being involved in fungal cross-feeding. Placed in an ecological context, strong antagonistic relationships of the mycoplankton community with other components of the plankton suggest that: (i) there is a top-down control by fungi on zooplankton and phytoplankton; (ii) fungi serve as a food source for zooplankton and thereby transfer nutrients and organic material; (iii) the dynamics of fungi harmful to other plankton groups are controlled by antagonistic fungal taxa.

The Plastisphere - Uncovering tightly attached plastic "specific" microorganisms.

In order to understand the degradation potential of plastics in the marine environment, microorganisms that preferentially colonize and interact with plastic surfaces, as opposed to generalists potentially colonising everything, need to be identified. Accordingly, it was hypothesized that i.) plastic "specific" microorganisms are closely attached to the polymeric surface and ii.) that specificity of plastics biofilms are rather related to members of the rare biosphere. To answer these hypotheses, a three phased experiment to stepwise uncover closely attached microbes was conducted. In Phase 1, nine chemically distinct plastic films and glass were incubated in situ for 21 months in a seawater flow through system. In Phase 2, a high-pressure water jet treatment technique was used to remove the upper biofilm layers to further, in Phase 3, enrich a plastic "specific" community. To proof whether microbes colonizing different plastics are distinct from each other and from other inert hard substrates, the bacterial communities of these different substrates were analysed using 16S rRNA gene tag sequencing. Our findings indicate that tightly attached microorganisms account to the rare biosphere and suggest the presence of plastic "specific" microorganisms/assemblages which could benefit from the given plastic properties or at least grow under limited carbon resources.

Mature biofilm communities on synthetic polymers in seawater - Specific or general?

To understand the ecological impacts of the "Plastisphere", those microbes need to be identified that preferentially colonize and interact with synthetic polymer surfaces, as opposed to general surface colonizers. It was hypothesized that the microbial biofilm composition varies distinctly between different substrates. A long-term incubation experiment was conducted (15month) with nine different synthetic polymer films as substrate as well as glass using a natural seawater flow-through system. To identify colonizing microorganisms, 16S and 18SrRNA gene tag sequencing was performed. The microbial biofilms of these diverse artificial surfaces were visualized via scanning electron microscopy. Biofilm communities attached to synthetic polymers are distinct from glass associated biofilms; apparently a more general marine biofilm core community serves as shared core among all synthetic polymers rather than a specific synthetic polymer community. Nevertheless, characteristic and discriminatory taxa of significantly different biofilm communities were identified, indicating their specificity to a given substrate.

The North Sea goes viral: Occurrence and distribution of North Sea bacteriophages.

Marine viruses are dominated by phages and have an enormous influence on microbial population dynamics, due to lysis and horizontal gene transfer. The aim of this study is to analyze the occurrence and diversity of phages in the North Sea, considering the virus-host interactions and biogeographic factors. The virus community of four sampling stations were described using virus metagenomics (viromes). The results show that the virus community was not evenly distributed throughout the North Sea. The dominant phage members were identified as unclassified phage group, followed by Caudovirales order. Myoviridae was the dominant phage family in the North Sea, which occurrence decreased from the coast to the open sea. In contrast, the occurrence of Podoviridae increased and the occurrence of Siphoviridae was low throughout the North Sea. The occurrence of other groups such as Phycodnaviridae decreased from the coast to the open sea. The coastal virus community was genetically more diverse than the open sea community. The influence of riverine inflow and currents, for instance the English Channel flow affects the genetic virus diversity with the community carrying genes from a variety of metabolic pathways and other functions. The present study offers the first insights in the virus community in the North Sea using viromes and shows the variation in virus diversity and the genetic information moved from coastal to open sea areas.

Bacterial community dynamics during the winter-spring transition in the North Sea.

Bacterioplankton dynamics at Helgoland Roads (54 degrees 11.3'N, 7 degrees 54.0'E) in the North Sea over the winter-spring transition were investigated. The bacterial community was analyzed and correlated with phytoplankton community data and abiotic parameters. The community structure was analyzed by ribosomal intergenic spacer analysis (RISA) and by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis. The linkage of abiotic and biotic environmental factors and bacterial community as well as phylotypes (sequenced DGGE bands) was analyzed by the ordination technique of canonical correspondence analysis (CCA). Generally, an influence of temperature and phytoplankton on the bacterial community during the sampling period was observed. Additionally, multivariate analysis by factors revealed an influence on specific bacterial phylotypes of these factors. Overall, results indicate that changes in the bacterial community were caused not only by abiotic factors but also by the phytoplankton community.

Comparative Omics and Trait Analyses of Marine Pseudoalteromonas Phages Advance the Phage OTU Concept.

Viruses influence the ecology and evolutionary trajectory of microbial communities. Yet our understanding of their roles in ecosystems is limited by the paucity of model systems available for hypothesis generation and testing. Further, virology is limited by the lack of a broadly accepted conceptual framework to classify viral diversity into evolutionary and ecologically cohesive units. Here, we introduce genomes, structural proteomes, and quantitative host range data for eight Pseudoalteromonas phages isolated from Helgoland (North Sea, Germany) and use these data to advance a genome-based viral operational taxonomic unit (OTU) definition. These viruses represent five new genera and inform 498 unaffiliated or unannotated protein clusters (PCs) from global virus metagenomes. In a comparison of previously sequenced Pseudoalteromonas phage isolates (n = 7) and predicted prophages (n = 31), the eight phages are unique. They share a genus with only one other isolate, Pseudoalteromonas podophage RIO-1 (East Sea, South Korea) and two Pseudoalteromonas prophages. Mass-spectrometry of purified viral particles identified 12-20 structural proteins per phage. When combined with 3-D structural predictions, these data led to the functional characterization of five previously unidentified major capsid proteins. Protein functional predictions revealed mechanisms for hijacking host metabolism and resources. Further, they uncovered a hybrid sipho-myovirus that encodes genes for Mu-like infection rarely described in ocean systems. Finally, we used these data to evaluate a recently introduced definition for virus populations that requires members of the same population to have >95% average nucleotide identity across at least 80% of their genes. Using physiological traits and genomics, we proposed a conceptual model for a viral OTU definition that captures evolutionarily cohesive and ecologically distinct units. In this trait-based framework, sensitive hosts are considered viral niches, while host ranges and infection efficiencies are tracked as viral traits. Quantitative host range assays revealed conserved traits within virus OTUs that break down between OTUs, suggesting the defined units capture niche and fitness differentiation. Together these analyses provide a foundation for model system-based hypothesis testing that will improve our understanding of marine copiotrophs, as well as phage-host interactions on the ocean particles and aggregates where Pseudoalteromonas thrive.