Recovering high-quality bacterial genomes from cross-contaminated cultures: a case study of marine Vibrio campbellii.

BACKGROUND: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. RESULTS: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. CONCLUSIONS: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

Annual recurrence of prokaryotic climax communities in shallow waters of the North Mediterranean.

In temperate coastal environments, wide fluctuations of biotic and abiotic factors drive microbiome dynamics. To link recurrent ecological patterns with planktonic microbial communities, we analysed a monthly-sampled 3-year time series of 16S rRNA amplicon sequencing data, alongside environmental variables, collected at two stations in the northern Adriatic Sea. Time series multivariate analyses allowed us to identify three stable, mature communities (climaxes), whose recurrence was mainly driven by changes in photoperiod and temperature. Mixotrophs (e.g., Ca. Nitrosopumilus, SUP05 clade, and Marine Group II) thrived under oligotrophic, low-light conditions, whereas copiotrophs (e.g., NS4 and NS5 clades) bloomed at higher temperatures and substrate availability. The early spring climax was characterised by a more diverse set of amplicon sequence variants, including copiotrophs associated with phytoplankton-derived organic matter degradation, and photo-auto/heterotrophic organisms (e.g., Synechococcus sp., Roseobacter clade), whose rhythmicity was linked to photoperiod lengthening. Through the identification of recurrent climax assemblages, we begin to delineate a typology of ecosystem based on microbiome composition and functionality, allowing for the intercomparison of microbial assemblages among different biomes, a still underachieved goal in the omics era.

Bacterial degradation of ctenophore Mnemiopsis leidyi organic matter.

Blooms of gelatinous zooplankton, an important source of protein-rich biomass in coastal waters, often collapse rapidly, releasing large amounts of labile detrital organic matter (OM) into the surrounding water. Although these blooms have the potential to cause major perturbations in the marine ecosystem, their effects on the microbial community and hence on the biogeochemical cycles have yet to be elucidated. We conducted microcosm experiments simulating the scenario experienced by coastal bacterial communities after the decay of a ctenophore (Mnemiopsis leidyi) bloom in the northern Adriatic Sea. Within 24 h, a rapid response of bacterial communities to the M. leidyi OM was observed, characterized by elevated bacterial biomass production and respiration rates. However, compared to our previous microcosm study of jellyfish (Aurelia aurita s.l.), M. leidyi OM degradation was characterized by significantly lower bacterial growth efficiency, meaning that the carbon stored in the OM was mostly respired. Combined metagenomic and metaproteomic analysis indicated that the degradation activity was mainly performed by Pseudoalteromonas, producing a large amount of proteolytic extracellular enzymes and exhibiting high metabolic activity. Interestingly, the reconstructed metagenome-assembled genome (MAG) of Pseudoalteromonas phenolica was almost identical (average nucleotide identity >99%) to the MAG previously reconstructed in our A. aurita microcosm study, despite the fundamental genetic and biochemical differences of the two gelatinous zooplankton species. Taken together, our data suggest that blooms of different gelatinous zooplankton are likely triggering a consistent response from natural bacterial communities, with specific bacterial lineages driving the remineralization of the gelatinous OM.IMPORTANCEJellyfish blooms are increasingly becoming a recurring seasonal event in marine ecosystems, characterized by a rapid build-up of gelatinous biomass that collapses rapidly. Although these blooms have the potential to cause major perturbations, their impact on marine microbial communities is largely unknown. We conducted an incubation experiment simulating a bloom of the ctenophore Mnemiopsis leidyi in the Northern Adriatic, where we investigated the bacterial response to the gelatinous biomass. We found that the bacterial communities actively degraded the gelatinous organic matter, and overall showed a striking similarity to the dynamics previously observed after a simulated bloom of the jellyfish Aurelia aurita s.l. In both cases, we found that a single bacterial species, Pseudoalteromonas phenolica, was responsible for most of the degradation activity. This suggests that blooms of different jellyfish are likely to trigger a consistent response from natural bacterial communities, with specific bacterial species driving the remineralization of gelatinous biomass.

The colonization of an irradiated environment: the case of microbial biofilm in a nuclear reactor.

The investigation of the microbial community change in the biofilm, growing on the walls of a containment tank of TRIGA nuclear reactor revealed a thriving community in an oligotrophic and heavy-metal-laden environment, periodically exposed to high pulses of ionizing radiation (IR). We observed a vertical IR resistance/tolerance stratification of microbial genera, with higher resistance and less diversity closer to the reactor core. One of the isolated Bacillus strains survived 15 kGy of combined gamma and proton radiation, which was surprising. It appears that there is a succession of genera that colonizes or re-colonizes new or IR-sterilized surfaces, led by Bacilli and/or Actinobacteria, upon which a photoautotrophic and diazotrophic community is established within a fortnight. The temporal progression of the biofilm community was evaluated also as a proxy for microbial response to radiological contamination events. This indicated there is a need for better dose-response models that could describe microbial response to contamination events. Overall, TRIGA nuclear reactor offers a unique insight into IR microbiology and provides useful means to study relevant microbial dose-thresholds during and after radiological contamination.

Inter-comparison of marine microbiome sampling protocols.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22-3 µm, 3-20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and "whole water" (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

Jellyfish detritus supports niche partitioning and metabolic interactions among pelagic marine bacteria.

BACKGROUND: Jellyfish blooms represent a significant but largely overlooked source of labile organic matter (jelly-OM) in the ocean, characterized by a high protein content. Decaying jellyfish are important carriers for carbon export to the ocean's interior. To accurately incorporate them into biogeochemical models, the interactions between microbes and jelly-OM have yet to be fully characterized. We conducted jelly-OM enrichment experiments in microcosms to simulate the scenario experienced by the coastal pelagic microbiome after the decay of a jellyfish bloom. We combined metagenomics, endo- and exo-metaproteomic approaches to obtain a mechanistic understanding on the metabolic network operated by the jelly-OM degrading bacterial consortium. RESULTS: Our analysis revealed that OM released during the decay of jellyfish blooms triggers a rapid shuffling of the taxonomic and functional profile of the pelagic bacterial community, resulting in a significant enrichment of protein/amino acid catabolism-related enzymes in the jelly-OM degrading community dominated by Pseudoalteromonadaceae, Alteromonadaceae and Vibrionaceae, compared to unamended control treatments. In accordance with the proteinaceous character of jelly-OM, Pseudoalteromonadaceae synthesized and excreted enzymes associated with proteolysis, while Alteromonadaceae contributed to extracellular hydrolysis of complex carbohydrates and organophosphorus compounds. In contrast, Vibrionaceae synthesized transporter proteins for peptides, amino acids and carbohydrates, exhibiting a cheater-type lifestyle, i.e. benefiting from public goods released by others. In the late stage of jelly-OM degradation, Rhodobacteraceae and Alteromonadaceae became dominant, growing on jelly-OM left-overs or bacterial debris, potentially contributing to the accumulation of dissolved organic nitrogen compounds and inorganic nutrients, following the decay of jellyfish blooms. CONCLUSIONS: Our findings indicate that specific chemical and metabolic fingerprints associated with decaying jellyfish blooms are substantially different to those previously associated with decaying phytoplankton blooms, potentially altering the functioning and biogeochemistry of marine systems. We show that decaying jellyfish blooms are associated with the enrichment in extracellular collagenolytic bacterial proteases, which could act as virulence factors in human and marine organisms' disease, with possible implications for marine ecosystem services. Our study also provides novel insights into niche partitioning and metabolic interactions among key jelly-OM degraders operating a complex metabolic network in a temporal cascade of biochemical reactions to degrade pulses of jellyfish-bloom-specific compounds in the water column. Video Abstract.

Bacterial Indicators Are Ubiquitous Members of Pelagic Microbiome in Anthropogenically Impacted Coastal Ecosystem.

Coastal zones are exposed to various anthropogenic impacts, such as different types of wastewater pollution, e.g., treated wastewater discharges, leakage from sewage systems, and agricultural and urban runoff. These various inputs can introduce allochthonous organic matter and microbes, including pathogens, into the coastal marine environment. The presence of fecal bacterial indicators in the coastal environment is usually monitored using traditional culture-based methods that, however, fail to detect their uncultured representatives. We have conducted a year-around in situ survey of the pelagic microbiome of the dynamic coastal ecosystem, subjected to different anthropogenic pressures to depict the seasonal and spatial dynamics of traditional and alternative fecal bacterial indicators. To provide an insight into the environmental conditions under which bacterial indicators thrive, a suite of environmental factors and bacterial community dynamics were analyzed concurrently. Analyses of 16S rRNA amplicon sequences revealed that the coastal microbiome was primarily structured by seasonal changes regardless of the distance from the wastewater pollution sources. On the other hand, fecal bacterial indicators were not affected by seasons and accounted for up to 34% of the sequence proportion for a given sample. Even more so, traditional fecal indicator bacteria (Enterobacteriaceae) and alternative wastewater-associated bacteria (Lachnospiraceae, Ruminococcaceae, Arcobacteraceae, Pseudomonadaceae and Vibrionaceae) were part of the core coastal microbiome, i.e., present at all sampling stations. Microbial source tracking and Lagrangian particle tracking, which we employed to assess the potential pollution source, revealed the importance of riverine water as a vector for transmission of allochthonous microbes into the marine system. Further phylogenetic analysis showed that the Arcobacteraceae in our data set was affiliated with the pathogenic Arcobacter cryaerophilus, suggesting that a potential exposure risk for bacterial pathogens in anthropogenically impacted coastal zones remains. We emphasize that molecular analyses combined with statistical and oceanographic models may provide new insights for environmental health assessment and reveal the potential source and presence of microbial indicators, which are otherwise overlooked by a cultivation approach.

Microbial Processing of Jellyfish Detritus in the Ocean.

When jellyfish blooms decay, sinking jellyfish detrital organic matter (jelly-OM), rich in proteins and characterized by a low C:N ratio, becomes a significant source of OM for marine microorganisms. Yet, the key players and the process of microbial jelly-OM degradation and the consequences for marine ecosystems remain unclear. We simulated the scenario potentially experienced by the coastal pelagic microbiome after the decay of a bloom of the cosmopolitan Aurelia aurita s.l. We show that about half of the jelly-OM is instantly available as dissolved organic matter and thus, exclusively and readily accessible to microbes. During a typical decay of an A. aurita bloom in the northern Adriatic Sea about 100 mg of jelly-OM L-1 becomes available, about 44 µmol L-1 as dissolved organic carbon (DOC), 13 µmol L-1 as total dissolved nitrogen, 11 µmol L-1 of total hydrolyzable dissolved amino acids (THDAA) and 0.6 µmol L-1 PO4 3-. The labile jelly-OM was degraded within 1.5 days (>98% of proteins, ∼70% of THDAA, 97% of dissolved free amino acids and the entire jelly-DOC pool) by a consortium of Pseudoalteromonas, Alteromonas, and Vibrio. These bacteria accounted for >90% of all metabolically active jelly-OM degraders, exhibiting high bacterial growth efficiencies. This implies that a major fraction of the detrital jelly-OM is rapidly incorporated into biomass by opportunistic bacteria. Microbial processing of jelly-OM resulted in the accumulation of tryptophan, dissolved combined amino acids and inorganic nutrients, with possible implications for biogeochemical cycles.

Jellyfish-Associated Microbiome in the Marine Environment: Exploring Its Biotechnological Potential.

Despite accumulating evidence of the importance of the jellyfish-associated microbiome to jellyfish, its potential relevance to blue biotechnology has only recently been recognized. In this review, we emphasize the biotechnological potential of host⁻microorganism systems and focus on gelatinous zooplankton as a host for the microbiome with biotechnological potential. The basic characteristics of jellyfish-associated microbial communities, the mechanisms underlying the jellyfish-microbe relationship, and the role/function of the jellyfish-associated microbiome and its biotechnological potential are reviewed. It appears that the jellyfish-associated microbiome is discrete from the microbial community in the ambient seawater, exhibiting a certain degree of specialization with some preferences for specific jellyfish taxa and for specific jellyfish populations, life stages, and body parts. In addition, different sampling approaches and methodologies to study the phylogenetic diversity of the jellyfish-associated microbiome are described and discussed. Finally, some general conclusions are drawn from the existing literature and future research directions are highlighted on the jellyfish-associated microbiome.

Bacteria associated with moon jellyfish during bloom and post-bloom periods in the Gulf of Trieste (northern Adriatic).

Jellyfish are a prominent component of the plankton community. They frequently form conspicuous blooms which may interfere with different human enterprises. Among the aspects that remain understudied are jellyfish associations with microorganisms having potentially important implications for organic matter cycling. To the best of our knowledge, this study is the first to investigate the bacterial community associated with live moon jellyfish (Aurelia solida, Scyohozoa) in the Adriatic Sea. Using 16S rRNA clone libraries and culture-based methods, we have analyzed the bacterial community composition of different body parts: the exumbrella surface, oral arms, and gastric cavity, and investigated possible differences in medusa-associated bacterial community structure at the time of the jellyfish population peak, and during the senescent phase at the end of bloom. Microbiota associated with moon jellyfish was different from ambient seawater bacterial assemblage and varied between different body parts. Betaproteobacteria (Burkholderia, Cupriavidus and Achromobacter) dominated community in the gastral cavity of medusa, while Alphaproteobacteria (Phaeobacter, Ruegeria) and Gammaproteobacteria (Stenotrophomonas, Alteromonas, Pseudoalteromonas and Vibrio) prevailed on 'outer' body parts. Bacterial community structure changed during senescent phase, at the end of the jellyfish bloom, showing an increased abundance of Gammaproteobacteria, exclusively Vibrio. The results of cultured bacterial isolates showed the dominance of Gammaproeteobacteria, especially Vibrio and Pseudoalteromonas in all body parts. Our results suggest that jellyfish associated bacterial community might have an important role for the host, and that anthropogenic pollution in the Gulf of Trieste might affect their community structure.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

Use of non-conventional yeast improves the wine aroma profile of Ribolla Gialla.

Consumer wine preferences are changing rapidly towards exotic flavours and tastes. In this work, we tested five non-conventional yeast strains for their potential to improve Ribolla Gialla wine quality. These strains were previously selected from numerous yeasts interesting as food production candidates. Sequential fermentation of Ribolla Gialla grape juice with the addition of the Saccharomyces cerevisiae T73 Lalvin industrial strain was performed. Zygosaccharomyces kombuchaensis CBS8849 and Kazachstania gamospora CBS10400 demonstrated positive organoleptic properties and suitable fermentation dynamics, rapid sugar consumption and industrial strain compatibility. At the same time, Torulaspora microellipsoides CBS6641, Dekkera bruxellensis CBS2796 and Dekkera anomala CBS77 were unsuitable for wine production because of poor fermentation dynamics, inefficient sugar consumption and ethanol production levels and major organoleptic defects. Thus, we selected strains of K. gamospora and Z. kombuchaensis that significantly improved the usually plain taste of Ribolla wine by providing additional aromatic complexity in a controlled and reproducible manner.

Jellyfish modulate bacterial dynamic and community structure.

Jellyfish blooms have increased in coastal areas around the world and the outbreaks have become longer and more frequent over the past few decades. The Mediterranean Sea is among the heavily affected regions and the common bloom-forming taxa are scyphozoans Aurelia aurita s.l., Pelagia noctiluca, and Rhizostoma pulmo. Jellyfish have few natural predators, therefore their carcasses at the termination of a bloom represent an organic-rich substrate that supports rapid bacterial growth, and may have a large impact on the surrounding environment. The focus of this study was to explore whether jellyfish substrate have an impact on bacterial community phylotype selection. We conducted in situ jellyfish-enrichment experiment with three different jellyfish species. Bacterial dynamic together with nutrients were monitored to assess decaying jellyfish-bacteria dynamics. Our results show that jellyfish biomass is characterized by protein rich organic matter, which is highly bioavailable to 'jellyfish-associated' and 'free-living' bacteria, and triggers rapid shifts in bacterial population dynamics and composition. Based on 16S rRNA clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis, we observed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria. The results of sequence analyses of bacterial isolates and of total bacterial community determined by culture independent genetic analysis showed the dominance of the Pseudoalteromonadaceae and the Vibrionaceae families. Elevated levels of dissolved proteins, dissolved organic and inorganic nutrient release, bacterial abundance and carbon production as well as ammonium concentrations characterized the degradation process. The biochemical composition of jellyfish species may influence changes in the amount of accumulated dissolved organic and inorganic nutrients. Our results can contribute insights into possible changes in bacterial population dynamics and nutrient pathways following jellyfish blooms which have important implications for ecology of coastal waters.

Deoxyribonucleoside kinases in two aquatic bacteria with high specificity for thymidine and deoxyadenosine.

Deoxyribonucleoside kinases (dNKs) are essential in the mammalian cell but their 'importance' in bacteria, especially aquatic ones, is less clear. We studied two aquatic bacteria, Gram-negative Flavobacterium psychrophilum JIP02/86 and Polaribacter sp. MED152, for their ability to salvage deoxyribonucleosides (dNs). Both had a Gram-positive-type thymidine kinase (TK1), which could phosphorylate thymidine, and one non-TK1 dNK, which could efficiently phosphorylate deoxyadenosine and slightly also deoxycytosine. Surprisingly, the four tested dNKs could not phosphorylate deoxyguanosine, and apparently, these two bacteria are missing this activity. When tens of available aquatic bacteria genomes were examined for the presence of dNKs, a majority had at least a TK1-like gene, but several lacked any dNKs. Apparently, among aquatic bacteria, the role of the dN salvage varies.

The global distribution and evolution of deoxyribonucleoside kinases in bacteria.

Deoxyribonucleoside kinases (dNKs) are important to DNA metabolism, especially in environments where nucleosides are freely available to be absorbed and used for the salvage pathway. Little has previously been known about the complement of dNKs in different bacterial genomes. However, it was believed that Gram-negative bacteria had a single dNK, while Gram-positive bacteria possessed several. An analysis of 992 fully sequenced bacterial genomes, including both Gram-positive and Gram-negative organisms, was conducted to investigate the phylogenetic relationship of all TK1-like and non-TK1-like dNKs. It was illustrated that both gene families evolved through a number of duplications and horizontal gene transfers, leading to the presence of multiple dNKs in different types of bacteria. The findings of this study provide a backbone for further studies into the evolution of the interplay between the de novo and salvage pathways in DNA synthesis with respect to environmental availability of deoxyribonucleosides and metabolic processes generating the provisions of different dNTPs.