A device for assessing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI).

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high-pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H-leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep-sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep-sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep-sea biotic processes.

Dynamics of the prokaryotic and eukaryotic microbial community during a cyanobacterial bloom.

Toxic cyanobacterial blooms frequently develop in eutrophic freshwater bodies worldwide. Microcystis species produce microcystins (MCs) as a cyanotoxin. Certain bacteria that harbor the mlr gene cluster, especially mlrA, are capable of degrading MCs. However, MC-degrading bacteria may possess or lack mlr genes (mlr+ and mlr- genotypes, respectively). In this study, we investigated the genotype that predominantly contributes to biodegradation and cyanobacterial predator community structure with change in total MC concentration in an aquatic environment. The 2 genotypes coexisted but mlr+ predominated, as indicated by the negative correlation between mlrA gene copy abundance and total MC concentration. At the highest MC concentrations, predation pressure by Phyllopoda, Copepoda, and Monogononta (rotifers) was reduced; thus, MCs may be toxic to cyanobacterial predators. The results suggest that cooperation between MC-degrading bacteria and predators may reduce Microcystis abundance and MC concentration.

Mesozooplankton taurine production and prokaryotic uptake in the northern Adriatic Sea.

Dissolved free taurine, an important osmolyte in phytoplankton and metazoans, has been shown to be a significant carbon and energy source for prokaryotes in the North Atlantic throughout the water column. However, the extent of the coupling between taurine production and consumption over a seasonal cycle has not been examined yet. We determined taurine production by abundant crustacean zooplankton and its role as a carbon and energy source for several prokaryotic taxa in the northern Adriatic Sea over a seasonal cycle. Taurine concentrations were generally in the low nanomolar range, reaching a maximum of 22 nmol L-1 in fall during a Pseudonitzschia bloom and coinciding with the highest zooplankton taurine release rates. Taurine accounted for up to 5% of the carbon, 11% of the nitrogen, and up to 71% of the sulfur requirements of heterotrophic prokaryotes. Members of the Roseobacter clade, Alteromonas, Thaumarchaeota, and Euryarchaeota exhibited higher cell-specific taurine assimilation rates than SAR11 cells. However, cell-specific taurine and leucine assimilation were highly variable in all taxa, suggesting species and/or ecotype specific utilization patterns of taurine and dissolved free amino acids. Copepods were able to cover the bulk taurine requirements of the prokaryotic communities in fall and winter and partly in the spring-summer period. Overall, our study emphasizes the significance of taurine as a carbon and energy source for the prokaryotic community in the northern Adriatic Sea and the importance of crustacean zooplankton as a significant source of taurine and other organic compounds for the heterotrophic prokaryotic community.

Substrate uptake patterns shape niche separation in marine prokaryotic microbiome.

Marine heterotrophic prokaryotes primarily take up ambient substrates using transporters. The patterns of transporters targeting particular substrates shape the ecological role of heterotrophic prokaryotes in marine organic matter cycles. Here, we report a size-fractionated pattern in the expression of prokaryotic transporters throughout the oceanic water column due to taxonomic variations, revealed by a multi-"omics" approach targeting ATP-binding cassette (ABC) transporters and TonB-dependent transporters (TBDTs). Substrate specificity analyses showed that marine SAR11, Rhodobacterales, and Oceanospirillales use ABC transporters to take up organic nitrogenous compounds in the free-living fraction, while Alteromonadales, Bacteroidetes, and Sphingomonadales use TBDTs for carbon-rich organic matter and metal chelates on particles. The expression of transporter proteins also supports distinct lifestyles of deep-sea prokaryotes. Our results suggest that transporter divergency in organic matter assimilation reflects a pronounced niche separation in the prokaryote-mediated organic matter cycles.

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.

A ubiquitous gammaproteobacterial clade dominates expression of sulfur oxidation genes across the mesopelagic ocean.

The deep ocean (>200 m depth) is the largest habitat on Earth. Recent evidence suggests sulfur oxidation could be a major energy source for deep ocean microbes. However, the global relevance and the identity of the major players in sulfur oxidation in the oxygenated deep-water column remain elusive. Here we combined single-cell genomics, community metagenomics, metatranscriptomics and single-cell activity measurements on samples collected beneath the Ross Ice Shelf in Antarctica to characterize a ubiquitous mixotrophic bacterial group (UBA868) that dominates expression of RuBisCO genes and of key sulfur oxidation genes. Further analyses of the gene libraries from the 'Tara Oceans' and 'Malaspina' expeditions confirmed the ubiquitous distribution and global relevance of this enigmatic group in the expression of sulfur oxidation and dissolved inorganic carbon fixation genes across the global mesopelagic ocean. Our study also underscores the unrecognized importance of mixotrophic microbes in the biogeochemical cycles of the deep ocean.

Limited carbon cycling due to high-pressure effects on the deep-sea microbiome.

Deep-sea microbial communities are exposed to high-pressure conditions, which has a variable impact on prokaryotes depending on whether they are piezophilic (that is, pressure-loving), piezotolerant or piezosensitive. While it has been suggested that elevated pressures lead to higher community-level metabolic rates, the response of these deep-sea microbial communities to the high-pressure conditions of the deep sea is poorly understood. Based on microbial activity measurements in the major oceanic basins using an in situ microbial incubator, we show that the bulk heterotrophic activity of prokaryotic communities becomes increasingly inhibited at higher hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was about one-third of that measured in the same community at atmospheric pressure conditions. In the bathypelagic zone-between 1,000 and 4,000 m depth-~85% of the prokaryotic community was piezotolerant and ~5% of the prokaryotic community was piezophilic. Despite piezosensitive-like prokaryotes comprising only ~10% (mainly members of Bacteroidetes, Alteromonas) of the deep-sea prokaryotic community, the more than 100-fold metabolic activity increase of these piezosensitive prokaryotes upon depressurization leads to high apparent bulk metabolic activity. Overall, the heterotrophic prokaryotic activity in the deep sea is likely to be substantially lower than hitherto assumed, with major impacts on the oceanic carbon cycling.

Autofluorescence Is a Common Trait in Different Oceanic Fungi.

Natural autofluorescence is a widespread phenomenon observed in different types of tissues and organisms. Depending on the origin of the autofluorescence, its intensity can provide insights on the physiological state of an organism. Fungal autofluorescence has been reported in terrestrial and human-derived fungal samples. Yet, despite the recently reported ubiquitous presence and importance of marine fungi in the ocean, the autofluorescence of pelagic fungi has never been examined. Here, we investigated the existence and intensity of autofluorescence in five different pelagic fungal isolates. Preliminary experiments of fungal autofluorescence at different growth stages and nutrient conditions were conducted, reflecting contrasting physiological states of the fungi. In addition, we analysed the effect of natural autofluorescence on co-staining with DAPI. We found that all the marine pelagic fungi that were studied exhibited autofluorescence. The intensity of fungal autofluorescence changed depending on the species and the excitation wavelength used. Furthermore, fungal autofluorescence varied depending on the growth stage and on the concentration of available nutrients. Collectively, our results indicate that marine fungi can be auto-fluorescent, although its intensity depends on the species and growth condition. Hence, oceanic fungal autofluorescence should be considered in future studies when fungal samples are stained with fluorescent probes (i.e., fluorescence in situ hybridization) since this could lead to misinterpretation of results.

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.

Response of microcystin biosynthesis and its biosynthesis gene cluster transcription in Microcystis aeruginosa on electrochemical oxidation.

Microcystin-LR (MC-LR), which is one of the most commonly found microcystins (MCs) in fresh water, has been proved to be a potential tumour promoter and classified as 2B by the International Agency for Research on Cancer. MC-LR decomposition and inhibition of MC-LR production in Microcystis aeruginosa were investigated under electrolysis condition using an electrolysis cell consisting of Ti/Pt electrodes and Nafion membrane. The relationship between the decrease in MC-LR concentration and transcription of MC-LR synthesis gene clusters was determined by performing real-time reverse transcription polymerase chain reaction (RT-qPCR) to monitor changes in the levels of transcription encoding mcyB and mcyD (cDNA to DNA) in M. aeruginosa NIES 1086 under electrolysis condition and three different conditions (i.e. oxygenated, air aerated and unaerated) as controls. Cell density decreased from day 2 under electrolysis than under the three controls. Intracellular MC-LR concentration was approximately 33 fg cell-1 under electrolysis from days 4 to 8, while those in the other conditions ranged in 40-50 fg cell-1. The mcyB transcription continuously decreased from day 2 to nondetectable level in day 6 under electrolysis, while this transcription was stabilised under the three controls. This result suggested that oxidative stress, such as hydroxyl radicals, played an important role in the down-regulation of mcyB and mcyD gene transcription level and the MC-LR concentration and cell density of M. aeruginosa.

The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity.

The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.

The specific delivery of proteins to human liver cells by engineered bio-nanocapsules.

A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice.

To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.

Polyamidoamine dendron-bearing lipid assemblies: their morphologies and gene transfection ability.

Assembly morphology made from lipids is controlled by the balance between the polar headgroup and the hydrophobic tails. In this study, we showed the various generations of polyamidoamine dendron-bearing lipids could form various assembly morphologies. Furthermore, the effect of the assembly morphologies made from dendron-bearing lipids for transfection abilities were examined. We synthesized three novel dendron-bearing lipids, DL-U2-G1 (G1), DL-U2-G2 (G2), and DL-U2-G3 (G3), which included first, second, and third generation polyamidoamine dendrons, respectively. Transmission electron microscopy showed that lipoplexes (complexes with cationic lipids and plasmid DNA) comprising G1 had multilamellar structures. G2 presented as aggregates of cubic particles and G3 exhibited clusters of spherical micelles. The ability to form complexes with plasmid DNA was in the decreasing order G3 > G2 > G1; calcein release from endosomes was in the order G3 > G2, G1; and transfection activity followed the order G1 > G2, G3. Interaction of the lipoplexes with heparin suggests that G3 had a lower level of plasmid DNA dissociation from lipoplexes than G1 in vitro. These results suggest that the size of the DL-U2 headgroup determines assembly morphology and that the structure markedly affects transfection activity.