Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant

Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters

“Super-blooms” of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species, were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystin-poisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine mammals due to cyanotoxins and confirms the existence of a novel class of marine “harmful algal bloom” in the Pacific coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from microcystin poisoning when consuming shellfish harvested at the land-sea interface

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean’s nitrogen inventory and primary productivity. Nitrogen-isotope data fromocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogenbudget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 TgNyr-1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method8 can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimatesN2-fixation rates by an averageof 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and c-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14+/-1TgNyr-1 and 24+/-1TgNyr-1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103+/-8TgNyr-1 to 177+/-8TgNyr-1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought

Widespread Distribution and Expression of Gamma A (UMB), an Uncultured, Diazotrophic, γ-Proteobacterial nifH Phylotype

Marine dinitrogen (N2) fixation studies have focused nearly exclusively on cyanobacterial diazotrophs; however γ-proteobacteria are an abundant component of the marine community and have been largely overlooked until recently. Here we present a phylogenetic analysis of all nifH γ-proteobacterial sequences available in public databases and qPCR data of a γ-proteobacterial phylotype, Gamma A (UMB), obtained during several research cruises. Our analysis revealed a complex diversity of diazotrophic γ-proteobacteria. One phylotype in particular, Gamma A, was described in several traditional and quantitative PCR studies. Though several γ-proteobacterial nifH sequences have been described as laboratory contaminants, Gamma A is part of a large cluster of sequences isolated from marine environments and distantly related to the clade of contaminants. Using a TaqMan probe and primer set, Gamma A nifH DNA abundance and expression were analyzed in nearly 1000 samples collected during 15 cruises to the Atlantic and Pacific Oceans. The data showed that Gamma A is an active, cosmopolitan diazotroph found throughout oxygenated, oligotrophic waters reaching maximum abundances of 8 × 104 nifH DNA copies l-1 and 5 × 105 nifH transcript copies l-1. Gamma A nifH transcript abundances were on average 3 fold higher than nifH DNA abundances. The widespread distribution and activity of Gamma A indicate that it has potential to be a globally important N2 fixing organism

Microbial life in volcanic lakes

Lakes in the craters of active volcanoes and their related streams are often characterised by conditions considered extreme for life, such as high temperatures, low pH and very high concentrations of dissolved metals and minerals. Such lakes tend to be transient features whose geochemistry can change markedly over short time periods. They might also vanish completely during eruption episodes or by drainage through the crater wall or floor. These lakes and their effluent streams and springs host taxonomically and metabolically diverse microorganisms belonging in the Archaea, Bacteria, and Eucarya. In volcanic ecosystems the relation between geosphere and biosphere is particularly tight; microbial community diversity is shaped by the geochemical parameters of the lake, and by the activities of microbes interacting with the water and sediments. Sampling these lakes is often challenging, and few have even been sampled once, especially in a microbiological context. Developments in high-throughput cultivation procedures, single-cell selection techniques, and massive increases in DNA sequencing throughput, should encourage efforts to define which microbes inhabit these features and how they interact with each other and the volcano. The study of microbial communities in volcanic lake systems sheds light on possible origins of life on early Earth. Other potential outcomes include the development of microbial inocula to promote plant growth in altered or degraded soils, bioremediation of contaminated waste or land, and the discovery of enzymes or other proteins industrial or medical applications

Small-scale carbon and nitrogen fluxes associated with Aphanizomenon sp. in the Baltic Sea.

Carbon and nitrogen fluxes in Aphanizomenon sp. colonies in the Baltic Sea were measured using a combination of microsensors, stable isotopes, mass spectrometry, and nanoscale secondary ion mass spectrometry (nanoSIMS). Cell numbers varied between 956 and 33 000 in colonies ranging in volume between 1.4 × 10−4 and 230 × 10−4 mm−3. The high cell content and their productivity resulted in steep O2 gradients at the colony–water interface as measured with an O2 microsensor. Colonies were highly autotrophic communities with few heterotrophic bacteria attached to the filaments. Volumetric gross photosynthesis in colonies was 78 nmol O2 mm−3 h−1. Net photosynthesis was 64 nmol O2 mm−3 h−1, and dark respiration was on average 15 nmol O2 mm−3 h−1 or 16% of gross photosynthesis. These volumetric photosynthesis rates belong to the highest measured in aquatic systems. The average cell-specific net carbon-fixation rate was 38 and 40 fmol C cell−1 h−1 measured by microsensors and by using stable isotopes in combination with mass spectrometry and nanoSIMS, respectively. In light, the net C:N fixation ratio of individual cells was 7.3±3.4. Transfer of fixed N2 from heterocysts to vegetative cells was fast, but up to 35% of the gross N2 fixation in light was released as ammonium into the surrounding water. Calculations based on a daily cycle showed a net C:N fixation ratio of 5.3. Only 16% of the bulk N2 fixation in dark was detected in Aphanizomenon sp. Hence, other organisms appeared to dominate N2 fixation and NH4+ release during darkness