Zinc stimulation of coastal productivity in low carbon dioxide environments.

Zinc (Zn) is a key micronutrient used by phytoplankton for carbon (C) acquisition, yet there have been few observations of its influence on natural oceanic phytoplankton populations. In this study, we observed Zn limitation of growth in the natural phytoplankton community of Terra Nova Bay, Antarctica, due to low (~220 µatm) pCO2 conditions, in addition to primary iron (Fe) limitation. Shipboard incubation experiments amended with Zn and Fe resulted in significantly higher chlorophyll a content and dissolved inorganic carbon drawdown compared to Fe addition alone. Zn and Fe response proteins detected in incubation and environmental biomass provided independent verification of algal co-stress for these micronutrients. These observations of Zn limitation under low pCO2 conditions demonstrate Zn can influence coastal primary productivity. Yet, as surface ocean pCO2 rises with continued anthropogenic emissions, the occurrence of Zn/C co-limitation will become rarer, impacting the biogeochemical cycling of Zn and other trace metal micronutrients.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. KEY POINTS: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production.

A Comparative Analysis of Mucus Immunomodulatory Properties from Seven Marine Gastropods from the Mediterranean Sea.

The treatment of inflammatory and immune-related diseases due to dysfunctioning of the immune system necessitates modulation of the immune response through immunomodulatory compounds. Marine environments are considered as a new frontier for health benefit product implementations. Marine biodiversity is still a low explored resource, despite it is expected to represent an important platform for chemical bioactive compounds. Within the phylum Mollusca, gastropods are known to synthetize mucus, the latter presenting relevant bioactive properties, e.g., related to immunomodulant molecules able to activate the innate and acquired immune system. This study proposes a bioprospecting of the immunomodulant activity of mucus isolated from seven common gastropod species from the Gulf of Naples (Mediterranean Sea). Results showed that not all mucus displayed a significant cytotoxic activity on the two human cancer cell lines A549 and A2058. On the other hand, the mucus from Bolinus brandaris was strongly bioactive and was therefore thoroughly investigated at cellular, molecular, and protein levels on the human monocytes U937 line. It can conclusively induce monocyte differentiation in vitro and significantly stimulate natural immunity response.

An optimised method for intact nuclei isolation from diatoms.

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.

Do plankton reflect the environmental quality status? The case of a post-industrial Mediterranean Bay.

While the effects of industrial contamination in coastal areas may persist for years in benthos communities, plankton should not show permanent impairments because of their high spatial dynamics, fast turnover times and pronounced seasonality. To test this hypothesis, in 2019 we conducted five surveys in the Bay of Pozzuoli (Gulf of Naples, Mediterranean Sea), in front of a dismissed steel factory and in the adjacent inshore coastal waters. High seasonal variability was observed for bacteria, phytoplankton and mesozooplankton, whereas plankton spatial gradients were relatively smooth during each survey. Plankton biomass and diversity did not reveal any effects of past industrial activities not even at the innermost stations of the Bay, which however showed some signals of present anthropogenic pressure. Hydrodynamic and morphological features likely play a prominent role in maintaining a relatively good status of the plankton of the Bay, which hints at the relevance of coastal circulation and meteorological dynamics to revitalize areas impacted by human activities.

Time resolved data unveils the complex DOM dynamics in a Mediterranean river.

In this study, dissolved organic carbon (DOC) data and optical properties (absorbance and fluorescence) of DOM, weekly collected in the Arno River for 2 years, are used to investigate the main processes determining DOM temporal dynamics in a small Mediterranean river, with torrential hydrology and medium-high human impact, and to quantify the contribution of this river to Med Sea carbon budget. A clear seasonal cycle of DOM, with DOC values ranging between 170 and 490 µM, was observed. Optical properties indicates that DOM quality in the river is different depending on the season; terrestrial humic-like substances prevail in winter, when discharge and floods are the main drivers of DOM concentration and quality, whereas autochthonous protein-like substances prevail in spring and summer, when biological processes dominate. Our results provide a robust estimate of the DOC flux to the Med Sea (9.6 · 109 g DOC yr-1) and of its range of variability (12.95 · 109-5.12 · 109 g DOC yr-1). The 80% of this flux was generally delivered during autumn/winter with significant amounts ascribed to single flood events (up to 26% in 2014). This study, by providing a rich dataset on water quantity and quality and by quantifying the importance of the hydrological regime on DOC transport, represents an important step toward a quantitative modeling of the Arno River.

Winter picoplankton diversity in an oligotrophic marginal sea.

Marine picoplankton, unicellular organisms with cell sizes up to 3 µm in diameter, numerically dominate marine ecosystems, encompassing Archaea, Bacteria, Eukarya (protists and fungi) as well as viruses. Autotrophic and heterotrophic picoplankton abundance and community composition with a focus on picoeukaryotes (PEs) were investigated in the winter of 2016 at three stations along a coast-to-offshore transect in the southern Adriatic Sea. Abundances were estimated by flow cytometry, while community composition by Illumina High-Throughput Sequencing (HTS) of 16S and 18S rRNA genes. The photosynthetic picoplankton diversity was also investigated by High-Performance Liquid Chromatography (HPLC) of liposoluble pigments. Heterotrophic bacteria and cyanobacteria (Prochlorococcus and Synechococcus) accounted for up to 7 × 105; 2.3 × 104 and 2.5 × 104 cells mL-1, respectively, while photosynthetic picoeukaryotes peaked with 3 × 103 cells mL-1. Prokaryotes, as revealed by HTS were dominated by Alphaproteobacteria (mainly SAR11, 44.91% of total 16S sequence reads), followed by Gammaproteobacteria (Oceanospirillales and Pseudomonadales, 14.96%), Bacteroidetes (mainly Flavobacteriales, 13%), Cyanobacteria (Prochlorococcus and Synechococcus, 9.52%), Marinimicrobia (SAR406, 7.97%), Deltaproteobacteria (SAR324, 3.83%), Actinobacteria (2.24%) and Chloroflexi (SAR202, 1.90%). Photosynthetic pigment concentrations were very low (12.12 µgL-1 at the most) and taxonomic pigments could be attributed to Prochlorococcus, Synechococcus, Prymnesiophyceae, Bacillariophyceae, Chrysophyceae, and Prasinophyceae. HTS data revealed that PEs were dominated by heterotrophs, such as Syndiniophyceae, parasitic dinoflagellates (79.67% of total 18S sequence reads), Dinophyceae (8.7%) and the radiolarians Collodaria belonging to Sphaerozoidae (22.1%) and Spumellaria (5.0%). On the other hand, photoautotrophs, including Chlorophyta (Mamiellophyceae, Prasinophyceae, Trebouxiophyceae, and Ulvophyceae), Stramenopiles (Bacillariophyta, Chrysophyceae, Dictyochophyceae, Pelagophyceae), photoautotrophic Cryptophyta and some Haptophyta (Prymnesiophyceae), did not exceed 5% of total sequence reads. This study provides the first snapshot of the PEs diversity in oligotrophic euphotic waters of the southern Adriatic Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin.

Distribution, Community Composition, and Potential Metabolic Activity of Bacterioplankton in an Urbanized Mediterranean Sea Coastal Zone.

Bacterioplankton are fundamental components of marine ecosystems and influence the entire biosphere by contributing to the global biogeochemical cycles of key elements. Yet, there is a significant gap in knowledge about their diversity and specific activities, as well as environmental factors that shape their community composition and function. Here, the distribution and diversity of surface bacterioplankton along the coastline of the Gulf of Naples (GON; Italy) were investigated using flow cytometry coupled with high-throughput sequencing of the 16S rRNA gene. Heterotrophic bacteria numerically dominated the bacterioplankton and comprised mainly Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes Distinct communities occupied river-influenced, coastal, and offshore sites, as indicated by Bray-Curtis dissimilarity, distance metric (UniFrac), linear discriminant analysis effect size (LEfSe), and multivariate analyses. The heterogeneity in diversity and community composition was mainly due to salinity and changes in environmental conditions across sites, as defined by nutrient and chlorophyll a concentrations. Bacterioplankton communities were composed of a few dominant taxa and a large proportion (92%) of rare taxa (here defined as operational taxonomic units [OTUs] accounting for <0.1% of the total sequence abundance), the majority of which were unique to each site. The relationship between 16S rRNA and the 16S rRNA gene, i.e., between potential metabolic activity and abundance, was positive for the whole community. However, analysis of individual OTUs revealed high rRNA-to-rRNA gene ratios for most (71.6% ± 16.7%) of the rare taxa, suggesting that these low-abundance organisms were potentially active and hence might be playing an important role in ecosystem diversity and functioning in the GON.IMPORTANCE The study of bacterioplankton in coastal zones is of critical importance, considering that these areas are highly productive and anthropogenically impacted. Their richness and evenness, as well as their potential activity, are very important to assess ecosystem health and functioning. Here, we investigated bacterial distribution, community composition, and potential metabolic activity in the GON, which is an ideal test site due to its heterogeneous environment characterized by a complex hydrodynamics and terrestrial inputs of varied quantities and quality. Our study demonstrates that bacterioplankton communities in this region are highly diverse and strongly regulated by a combination of different environmental factors leading to their heterogeneous distribution, with the rare taxa contributing to a major proportion of diversity and shifts in community composition and potentially holding a key role in ecosystem functioning.

Toxicity of diatom polyunsaturated aldehydes to marine bacterial isolates reveals their mode of action.

Diatoms produce and release polyunsaturated aldehydes (PUAs) during senescence in culture and at the end of blooms in nature and these compounds play different ecological roles, as infochemicals, allelochemicals and pheromones In order to elucidate the toxic effects of PUAs, we isolated six bacterial strains from the Mediterranean Sea during a diatom bloom and tested their tolerance to PUA in terms of growth and cell membrane properties. Based upon 16S rRNA sequencing, these bacteria were assigned to the genera Pseudomonas, Sufflavibacter, Halomonas, Vibrio, Idiomarina, and Labrenzia. Growth of these strains was reduced by 50% (EC50) at PUA concentrations ranging from 600 to 1700 µM of 2E,4E/Z-heptadienal (HEPTA), 400-800 µM of 2E, 4E/Z-octadienal (OCTA), and 70-400 µM of 2E, 4E/Z-decadienal (DECA). Two of these strains, Vibrio sp. and Halomonas, sp. were also investigated for membrane fatty acid composition in terms of adaptive modifications of their degree of saturation (ratio between saturated and unsaturated fatty acids) by GC-FID. A direct correlation between hydrophobicity and PUA toxicity was observed, and these bacteria were also found to react to PUAs by increasing the degree of saturation of their membranes fatty acids. Tested PUAs were 4-fold more toxic than the well-investigated n-alkanols, most probably due to their additional chemical aldehyde toxicity to disrupting proteins by the formation of Schiff's bases, and therefore, they act as very toxic and effective poison, probably accumulating in cytoplasmic membranes because of their high hydrophobicity.

Taxonomic and functional diversity of a coastal planktonic bacterial community in a river-influenced marine area.

The Gulf of Naples is a dynamical area with intense exchanges between offshore oligotrophic and coastal eutrophic waters with frequent freshwater inputs. The Sarno River, one of the most polluted rivers in Europe, strongly contributes to the pollution of the area, discharging high amounts of heavy metals and organic wastes from heavily cultivated and industrial areas. This paper reports on the diversity and community structure of the marine residential Bacteria and Archaea of the Gulf of Naples in an area close to the river Sarno plume and investigates their small-scale taxonomic diversity and expression patterns as a proxy of potential metabolic activity using metagenomics and metatranscriptomics. Bacteria and Archaea were mainly represented by marine clades, with only minor contributors from freshwater ones. The community was dominated by Alpha- and Gammaproteobacteria, of which Rhodospirillales, Pelagibacteriales, and Oceanospirilalles were most represented. However, Alteromonadales and Rhodobacterales were the most active, despite their relative lower abundance, suggesting that they are important for overall ecosystem functioning and nutrient cycling. Nitrification and a reversed form of dissimilatory sulfate reduction were the major metabolic processes found in the metatrascriptomes and were mainly associated to Nitrosopumilales and Pelagibacter, respectively. No clear indication of transcripts related to stress induced by heavy metals or organic pollutants was found. In general, despite the high loads of pollutants discharged continuously by the Sarno River, the microbial community did not show marks of stress-induced changes neither structural nor functional, thus suggesting that this river has little or no effect on the planktonic bacterial community of the Gulf of Naples.

A Bacillus sp. isolated from sediments of the Sarno River mouth, Gulf of Naples (Italy) produces a biofilm biosorbing Pb(II).

A Pb-resistant bacterial strain (named hereinafter Pb15) has been isolated from highly polluted marine sediments at the Sarno River mouth, Italy, using an enrichment culture to which Pb(II) 0.48mmoll(-1) were added. 16S rRNA gene sequencing (Sanger) allowed assignment of the isolate to the genus Bacillus, with Bacillus pumilus as the closest species. The isolate is resistant to Pb(II) with a minimum inhibitory concentration (MIC) of 4.8mmoll(-1) and is also resistant to Cd(II) and Mn(II) with MIC of 2.22mmoll(-1) and 18.20mmoll(-1), respectively. Inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that Pb inoculated in the growth medium is absorbed by the bacterial cells at removal efficiencies of 31.02% and 28.21% in the presence of 0.48mmoll(-1) or 1.20mmoll(-1) Pb(II), respectively. Strain Pb15 forms a brown and compact biofilm when grown in presence of Pb(II). Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-ray Spectroscopy (SEM-EDS) confirm that the biofilm contains Pb, suggesting an active biosorption of this metal by the bacterial cells, sequestering 14% of inoculated Pb as evidenced by microscopic analyses. Altogether, these observations support evidence that strain Pb15 has potentials for being used in bioremediation of its native polluted sediments, with engineering solutions to be found in order to eliminate the adsorbed Pb before replacement of sediments in situ.

Organic-matter degradative potential of Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica).

Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica) during austral summer 2003 was phenotypically characterized and its capability of degrading organic matter was tested. We evaluated specific bacterial growth rates (mu) to understand at which temperatures bacterial growth shows a linear and direct relationship with the available substrate (4-22 degrees C) and afterwards we tested H. glaciei growth curves and degradative potential at 0, 10 and 37 degrees C using two different media (one enriched and one depleted in PO(4)). The strain grew exponentially only at 10 degrees C. The fastest hydrolysis rates were expressed by enzymes aimed at polysaccharide degradation (alpha-D-glucosidase, beta-D-glucosidase and beta-D-galactosidase) while alkaline phosphatase and aminopeptidase activities were rather low. Our data suggest a preferential demand for carbon derived from carbohydrates rather than from proteins: ectoenzyme activities transformed into carbon mobilization from organic polymers, showed that the total carbon potentially released from polysaccharides can be almost one order of magnitude higher than the protein carbon mobilization. Principal component analysis of the enzyme affinity separated the six experimental conditions, highlighting how different physical (temperature) and chemical (PO(4) enrichment or depletion) features actively lead to a differentiation in the efficiency of the ectoenzymes produced, resulting in preferential degradation of diverse kinds of organic substrates.