Non-cyanobacterial diazotrophs: global diversity, distribution, ecophysiology, and activity in marine waters.

Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research.

The microbiome of a bacterivorous marine choanoflagellate contains a resource-demanding obligate bacterial associate.

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete 'Comchoano' genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella's that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

Alphaproteobacteria facilitate Trichodesmium community trimethylamine utilization.

In the surface waters of the warm oligotrophic ocean, filaments and aggregated colonies of the nitrogen (N)-fixing cyanobacterium Trichodesmium create microscale nutrient-rich oases. These hotspots fuel primary productivity and harbour a diverse consortium of heterotrophs. Interactions with associated microbiota can affect the physiology of Trichodesmium, often in ways that have been predicted to support its growth. Recently, it was found that trimethylamine (TMA), a globally abundant organic N compound, inhibits N2 fixation in cultures of Trichodesmium without impairing growth rate, suggesting that Trichodesmium can use TMA as an alternate N source. In this study, 15 N-TMA DNA stable isotope probing (SIP) of a Trichodesmium enrichment was employed to further investigate TMA metabolism and determine whether TMA-N is incorporated directly or secondarily via cross-feeding facilitated by microbial associates. Herein, we identify two members of the marine Roseobacter clade (MRC) of Alphaproteobacteria as the likely metabolizers of TMA and provide genomic evidence that they converted TMA into a more readily available form of N, e.g., ammonium (NH4 + ), which was subsequently used by Trichodesmium and the rest of the community. The results implicate microbiome-mediated carbon (C) and N transformations in modulating N2 fixation and thus highlight the involvement of host-associated heterotrophs in global biogeochemical cycling.

Metagenomics and Quantitative Stable Isotope Probing Offer Insights into Metabolism of Polycyclic Aromatic Hydrocarbon Degraders in Chronically Polluted Seawater.

Bacterial biodegradation is a significant contributor to remineralization of polycyclic aromatic hydrocarbons (PAHs)-toxic and recalcitrant components of crude oil as well as by-products of partial combustion chronically introduced into seawater via atmospheric deposition. The Deepwater Horizon oil spill demonstrated the speed at which a seed PAH-degrading community maintained by chronic inputs responds to acute pollution. We investigated the diversity and functional potential of a similar seed community in the chronically polluted Port of Los Angeles (POLA), using stable isotope probing with naphthalene, deep-sequenced metagenomes, and carbon incorporation rate measurements at the port and in two sites in the San Pedro Channel. We demonstrate the ability of the community of degraders at the POLA to incorporate carbon from naphthalene, leading to a quick shift in microbial community composition to be dominated by the normally rare Colwellia and Cycloclasticus We show that metagenome-assembled genomes (MAGs) belonged to these naphthalene degraders by matching their 16S-rRNA gene with experimental stable isotope probing data. Surprisingly, we did not find a full PAH degradation pathway in those genomes, even when combining genes from the entire microbial community, leading us to hypothesize that promiscuous dehydrogenases replace canonical naphthalene degradation enzymes in this site. We compared metabolic pathways identified in 29 genomes whose abundance increased in the presence of naphthalene to generate genomic-based recommendations for future optimization of PAH bioremediation at the POLA, e.g., ammonium as opposed to urea, heme or hemoproteins as an iron source, and polar amino acids.IMPORTANCE Oil spills in the marine environment have a devastating effect on marine life and biogeochemical cycles through bioaccumulation of toxic hydrocarbons and oxygen depletion by hydrocarbon-degrading bacteria. Oil-degrading bacteria occur naturally in the ocean, especially where they are supported by chronic inputs of oil or other organic carbon sources, and have a significant role in degradation of oil spills. Polycyclic aromatic hydrocarbons are the most persistent and toxic component of crude oil. Therefore, the bacteria that can break those molecules down are of particular importance. We identified such bacteria at the Port of Los Angeles (POLA), one of the busiest ports worldwide, and characterized their metabolic capabilities. We propose chemical targets based on those analyses to stimulate the activity of these bacteria in case of an oil spill in the Port POLA.

Direct Utilization of Organic Nitrogen by Phytoplankton and Its Role in Nitrogen Cycling Within the Southern California Bight.

The new production model attempts to quantify the amount of organic material exported from surface waters based on the form of nitrogen (N) being utilized. Dissolved organic N (DON) is rarely assessed during such investigations and even less is understood about the organisms involved in these different transformations within the complex N cycle. Stable isotope probing (SIP) and uptake activity measurements were combined to investigate the dynamics of new and regenerated production during the spring within the Southern California Bight (SCB). We examined the uptake and assimilation of several nitrogenous substrates at several depths to quantify these processes and identify the active communities across all three domains of life that are driving each transformation. Several reoccurring members closely related to the eukaryotic diatom Chaetoceros, dominated assimilation of NO3 - and urea through the water column, and contributed greatly to the overall production. Prokaryotic growth was predominantly carried out through NH4 + assimilation with transitions from Flavobacteria to Rhodobacteraceae and Marine Group II Euryarchaeota to Marine Group I Thaumarchaeota with increasing depth for bacterial and archaeal clades, respectively. Only urea uptake and SIP activity correlated with each other, likely demonstrating that cellular transport and incorporation of urea were coupled. SIP was therefore able to identify the organisms primarily responsible for urea cycling at each depth during this investigation. The role of diatoms within high nutrient areas are well defined but their part in DON cycling in highly stratified regimes is less well understood. Here we demonstrate their ability to efficiently scavenge urea in situ, allowing certain diatoms to outcompete the rest of the community. This diversion of DON away from the trophically inefficient microbial loop directly back into the larger, particle forming populations would alter the current view of microbial food webs. This proposed "phytoplankton shunt" of organic material could potentially enhance the biological pump by mitigating losses due to trophic transfers while increasing DON flux due to ballasting. Our results provide unique biogeochemical and ecological insight into the dynamics and diversity of N cycling and the organisms involved within the surface waters of the SCB.

Diazotrophic Macroalgal Associations With Living and Decomposing Sargassum.

Despite several studies reporting diazotrophic macroalgal associations (DMAs), biological nitrogen fixation (BNF) is still largely overlooked as a potential source of nitrogen (N) for macroalgal productivity. We investigated the role of BNF, via the acetylene reduction method, throughout different life stages of the invasive macroalga, Sargassum horneri, in its non-native Southern California coastal ecosystem. Throughout most of its life cycle, BNF rates were not detectable or yielded insignificant amounts of fixed N to support S. horneri productivity. However, during late summer when nutrient concentrations are usually at their minimum, BNF associated with juvenile S. horneri contributed ∼3-36% of its required N, potentially providing it with a competitive advantage. As DMAs remain poorly understood within macroalgal detrital systems, long term (15-28 days) laboratory decomposition time series were carried out to investigate the role of BNF throughout decomposition of the endemic macroalga, S. palmeri, and the invasive S. horneri. Nitrogenase activity increased drastically during the second phase of decomposition, when increasing microbial populations are typically thought to drive macroalgal degradation, with BNF rates associated with S. palmeri and S. horneri reaching up to 65 and 247 nmol N × g-1(dw) × h-1, respectively. Stimulation of BNF rates by glucose and mannitol additions, up to 42× higher rates observed with S. palmeri, suggest that labile carbon may be limiting at varying degrees in these detrital systems. Comparable, if not higher, dark BNF rates relative to light incubations during S. horneri decomposition suggest an important contribution from heterotrophic N fixers. Inhibition of nitrogenase activity, up to 98%, by sodium molybdate additions also suggest that sulfate reducers may be an important constituent of the detrital diazotrophic community. As labile N can become limiting to the microbial community during macroalgal decomposition, our results suggest that BNF may provide a source of new N, alleviating this limitation. Additionally, while BNF is rarely considered as a source for N enrichment with aging macroalgal detritus, we found it to account for ∼1-11% of N immobilized with decaying S. horneri. Our investigations suggest that DMAs may be globally important with Sargassum and potentially occur within other macroalgal detrital systems.

Intraclade Heterogeneity in Nitrogen Utilization by Marine Prokaryotes Revealed Using Stable Isotope Probing Coupled with Tag Sequencing (Tag-SIP).

Nitrogen can greatly influence the structure and productivity of microbial communities through its relative availability and form. However, the roles of specific organisms in the uptake of different nitrogen species remain poorly characterized. Most studies seeking to identify agents of assimilation have been correlative, indirectly linking activity measurements (e.g., nitrate uptake) with the presence or absence of biological markers, particularly functional genes and their transcripts. Evidence is accumulating of previously underappreciated functional diversity in major microbial subpopulations, which may confer physiological advantages under certain environmental conditions leading to ecotype divergence. This microdiversity further complicates our view of genetic variation in environmental samples requiring the development of more targeted approaches. Here, next-generation tag sequencing was successfully coupled with stable isotope probing (Tag-SIP) to assess the ability of individual phylotypes to assimilate a specific N source. Our results provide the first direct evidence of nitrate utilization by organisms thought to lack the genes required for this process including the heterotrophic clades SAR11 and the Archaeal Marine Group II. Alternatively, this may suggest the existence of tightly coupled metabolisms with primary assimilators, e.g., symbiosis, or the rapid and efficient scavenging of recently released products by highly active individuals. These results may be connected with global dominance often seen with these clades, likely conferring an advantage over other clades unable to access these resources. We also provide new direct evidence of in situ nitrate utilization by the cyanobacterium Prochlorococcus in support of recent findings. Furthermore, these results revealed widespread functional heterogeneity, i.e., different levels of nitrogen assimilation within clades, likely reflecting niche partitioning by ecotypes.

Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish.

Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1)). Fluoxetine treatments of 25 and 50 microgg(-1) were sub-lethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25 microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.

Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish.

Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1). Fluoxetine treatments of 25 and 50 microgg(-1) were sublethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25g microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.