SamplingIn order to compare the commonly used 15 N 2 tracer addition method to measure N 2 fixation 1 with the addition of 15 N 2 -enriched water as suggested by Mohr et al. (2010) 2 , seawater was sampled on two cruises in the Atlantic Ocean, the first on board R/V Meteor (M80/1) on a longitudinal transect (23°W) between 15°N and 5°S, the second on board R/V Polarstern (ANT-XXVI/1) on a transect between 54°N and 54°S (Bremerhaven, Germany to Punta Arenas, Chile). In total 39 triplicate incubations were conducted with both methods in parallel. On the M80/1 cruise, seawater was sampled at 11 stations from the surface (bucket), 20 m depth and the chlorophyll maximum (CTD rosette sampler) at 7:00 in the morning, whereas on the ANT-XXVI/1 cruise, seawater was sampled at 6 stations at 16:00 from the ship's clean seawater supply which is installed at 11 m depth (keel of the ship). IncubationsSeawater samples were filled headspace-free (bubble addition method) or with a 100-150 ml headspace (dissolved method) into 4.5 L polycarbonate bottles and closed with Teflon ® -coated butyl rubber septum caps. To determine N 2 fixation rates with the bubble-addition method, a 4.5 mL 15 N 2 gas bubble (Sigma-Aldrich, ≥98 atom%) was injected through the septa into each of triplicate bottles (yielding a theoretical enrichment of ~12 atom% assuming a rapid isotopic equilibration between the added 15 N 2 gas and the ambient dissolved N 2 of the water sample). After injection, bottles were gently inverted one hundred times. For comparison of N 2 fixation rates, we added 15 N 2 -enriched seawater to a second set of triplicate bottles (dissolution method). In detail, the preparation of the 15 N 2 -enriched seawater was started by degassing filtered seawater (0.2 µm filtered, Durapore) using a membrane flowthrough system (Mini-Module, Membrana) in which the seawater flowed on the inside of the membrane and a vacuum (-960 mbar, water jet pump) was applied to the outer side of the membrane. The seawater flow rate was about 400 -500 mL min -1 and seawater was recirculated for the first 10-15 min of the degassing step. Degassed seawater was then filled directly from the flow-through system into evacuated gas-tight 3L Tedlar® bags without a headspace. Addition of 15 N 2 gas was dependent on the amount of seawater in the Tedlar® bag and was added at a ratio of 10 ml 15 N 2 per 1L seawater. The volume of degassed seawater in SUPPLEMENTARY INFORMATION

Microbial dinitrogen (N) fixation, the nitrogenase enzyme-catalysed reduction of N gas into biologically available ammonia, is the main source of new nitrogen (N) in the ocean. For more than 50 years, oceanic N fixation has mainly been attributed to the activity of the colonial cyanobacterium Trichodesmium. Other smaller N-fixing microorganisms (diazotrophs)-in particular the unicellular cyanobacteria group A (UCYN-A)-are, however, abundant enough to potentially contribute significantly to N fixation in the surface waters of the oceans. Despite their abundance, the contribution of UCYN-A to oceanic N fixation has so far not been directly quantified. Here, we show that in one of the main areas of oceanic N fixation, the tropical North Atlantic, the symbiotic cyanobacterium UCYN-A contributed to N fixation similarly to Trichodesmium. Two types of UCYN-A, UCYN-A1 and -A2, were observed to live in symbioses with specific eukaryotic algae. Single-cell analyses showed that both algae-UCYN-A symbioses actively fixed N, contributing ∼20% to N fixation in the tropical North Atlantic, revealing their significance in this region. These symbioses had growth rates five to ten times higher than Trichodesmium, implying a rapid transfer of UCYN-A-fixed N into the food web that might significantly raise their actual contribution to N fixation. Our analysis of global 16S rRNA gene databases showed that UCYN-A occurs in surface waters from the Arctic to the Antarctic Circle and thus probably contributes to N fixation in a much larger oceanic area than previously thought. Based on their high rates of N fixation and cosmopolitan distribution, we hypothesize that UCYN-A plays a major, but currently overlooked role in the oceanic N cycle.

Marine macroalgae are constantly exposed to epibacterial colonizers. The epiphytic bacterial patterns and their temporal and spatial variability on host algae are poorly understood. To investigate the interaction between marine macroalgae and epiphytic bacteria, this study tested if the composition of epibacterial communities on different macroalgae was specific and persisted under varying biotic and abiotic environmental conditions over a 2-year observation time frame. Epibacterial communities on the co-occurring macroalgae Fucus vesiculosus, Gracilaria vermiculophylla and Ulva intestinalis were repeatedly sampled in summer and winter of 2007 and 2008. The epibacterial community composition was analysed by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene libraries. Epibacterial community profiles did not only differ significantly at each sampling interval among algal species, but also showed consistent seasonal differences on each algal species at a bacterial phylum level. These compositional patterns re-occurred at the same season of two consecutive years. Within replicates of the same algal species, the composition of bacterial phyla was subject to shifts at the bacterial species level, both within the same season but at different years and between different seasons. However, 7-16% of sequences were identified as species specific to the host alga. These findings demonstrate that marine macroalgae harbour species-specific and temporally adapted epiphytic bacterial biofilms on their surfaces. Since several algal host-specific bacteria were highly similar to other bacteria known to either avoid subsequent colonization by eukaryotic larvae or to exhibit potent antibacterial activities, algal host-specific bacterial associations are expected to play an important role for marine macroalgae.

Methanosarcina mazei and related mesophilic archaea are the only organisms fermenting acetate, methylamines, and methanol to methane and carbon dioxide, contributing significantly to greenhouse gas production. The biochemistry of these metabolic processes is well studied, and genome sequences are available, yet little is known about the overall transcriptional organization and the noncoding regions representing 25% of the 4.01-Mb genome of M. mazei. We present a genome-wide analysis of transcription start sites (TSS) in M. mazei grown under different nitrogen availabilities. Pyrosequencing-based differential analysis of primary vs. processed 5 ends of transcripts discovered 876 TSS across the M. mazei genome. Unlike in other archaea, in which leaderless mRNAs are prevalent, the majority of the detected mRNAs in M. mazei carry long untranslated 5 regions. Our experimental data predict a total of 208 small RNA (sRNA) candidates, mostly from intergenic regions but also antisense to 5 and 3 regions of mRNAs. In addition, 40 new small mRNAs with ORFs of <30 aa were identified, some of which might have dual functions as mRNA and regulatory sRNA. We confirmed differential expression of several sRNA genes in response to nitrogen availability. Inspection of their promoter regions revealed a unique conserved sequence motif associated with nitrogen-responsive regulation, which might serve as a regulator binding site upstream of the common IIB recognition element. Strikingly, several sRNAs antisense to mRNAs encoding transposases indicate nitrogen-dependent transposition events. This global TSS map in archaea will facilitate a better understanding of transcriptional and posttranscriptional control in the third domain of life.Methanosarcina mazei strain Gö1 is a representative methaneproducing archaeon of ecologic significance because of its role in biogenic methane production in various anaerobic habitats on Earth (1). The genome sequences of M. mazei and its close relatives Methanosarcina acetivorans and Methanosarcina barkeri have recently become available and have revealed an unexpected low proportion of coding region (74.2% in M. acetivorans, 75.15% in M. mazei, and 79.2% in M. barkeri) (2-4). The biochemical basis of methanogenesis has been analyzed in considerable detail (5, 6). In contrast, little is known about global regulatory networks that ensure survival in periods of nutrient starvation or stress in this important group of archaea. More than 50 predicted transcriptional regulators were annotated in the genome of M. mazei. Strikingly, most of them seem to be closely related to bacterial proteins (2), whereas the basic components of the archaeal transcription and translation machineries generally are more similar to those of eukaryotes (7). A recent genetic study (8) discovered the first global transcriptional regulator of M. mazei, the nitrogen regulator NrpR, which was experimentally demonstrated to globally repress transcription of nitrogen fixation and assimilation genes in response to the nitrogen source.Bes...

No abstract