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 harbor 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 receives nitrogen from TMA. In this study, 15N-TMA DNA stable isotope probing (SIP) of a Trichodesmium enrichment was employed to further investigate TMA metabolism and determine if 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., 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.

erythraeum metabolizes TMA through a novel biochemical pathway, it must receive 1 9 9 TMA-N in an alternate form, i.e., following TMA catabolism by associated microbiota.
2 0 0 In this study, SIP was employed to determine if TMA-N was incorporated 2 0 1 directly or secondarily via cross-feeding facilitated by the Trichodesmium consortium. bioavailable form that was subsequently used by the rest of the community. Additionally, we found genomic evidence that these MRC taxa have the metabolic 2 0 6 potential to catabolize TMA, thereby transforming TMA nitrogen to ammonium, 2 0 7 further demonstrating the potential for cross-feeding over time (Figure 3 and 4). of the MRC may play an important role in Trichodesmium ecology and physiology. Genes for TMA and TMAO metabolism are common but not universal among 2 2 1 the MRC (Chen, 2012;Lidbury et al., 2014) and enable some taxa to use TMA as a conversion of TMA to a more accessible nitrogen species, such as ammonium, as is catabolism and release ammonium, which then serves as an N source for 2 5 0 Trichodesmium and the rest of the community. In this scenario, the rapid inhibition of 2 5 1 N 2 fixation in Trichodesmium apparent on day 1 (Figure 2) is explained by MRC 2 5 2 ammonium production.

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While the results support this model, they do not rule out the possibility that Trichodesmium is also capable of TMA metabolism. It is possible that both MRC and 2 5 5 Trichodesmium utilize TMA (i.e., Figure 1, Option C) and that we saw MRC utilize TMA directly, it would need to do so by a yet unknown pathway, since T. from each culture daily to monitor N 2 fixation and growth rate, as well as to collect 3 1 5 biomass for DNA extraction. Nitrogenase activity was quantified by the acetylene reduction assay  This assay leverages the nitrogenase enzyme's ability to reduce triple-bonded 3 2 1 acetylene gas (C 2 H 2 ) to double-bonded ethylene gas (C 2 H 4 ), both of which can be injected with acetylene gas, after which ethylene production is measured over time and used as a proxy for calculating the sample's ability to reduce nitrogen gas (N 2 ) to 3 2 5 ammonium (NH 4 + ).

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In this study, acetylene gas was generated by mixing 15.0 g of calcium and Porapak R column. Peak height readings were integrated using PeakSimple  Culture growth was monitored via relative fluorescence of chlorophyll A, 3 4 8 measured in vivo using a Trilogy Laboratory Fluorometer (Turner Designs).

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Following the conclusion of the daily acetylene reduction assay, in vitro chlorophyll  Subsamples were collected from each culture daily for DNA extraction - a DNeasy PowerSoil Pro kit (Qiagen) following the manufacturer's protocol. Coulter) and spun for 72 hours at 136,000 × g av and 20°C. Tubes were then 3 6 8 fractionated in ~100 µl increments via displacement with mineral oil. Fraction densities were calculated using an AR200 Digital Refractometer (Reichert Technologies). DNA was precipitated from each fraction with PEG-NaCl (30% PEG, Exact sequence variants were inferred from raw reads following the Deblur reads generated with the 515F-Y / 926R primer pair are non-overlapping, and, 3 9 7 1 7 therefore, could not be merged. Instead, 18S forward reads were trimmed to 220 3 9 8 bases, reverse reads were trimmed to 179 bases, and the two were concatenated.  remove ASVs that constituted less than 0.01% of the total reads for each treatment, ASV enrichment on day 1 (Figure 3). Since 15 N incorporation cannot be less than 0, DNA extraction was carried out as described above. DNA was sent to  sequences 100% identical to Seq13 and Seq14, respectively, were identified using   Colonies of marine cyanobacteria Trichodesmium interact with associated 5 0 9 bacteria to acquire iron from dust. Commun Biol 2: 1-8. with QIIME 2's q2-feature-classifier plugin. Microbiome 6: 90. Ocean. Global Biogeochem Cycles 19:.  'omics data. PeerJ 3: e1319. Trichodesmium microbiome can modulate host N2 fixation. Limnol Oceanogr Lett 3: 401-408.   Microbiome of Trichodesmium Colonies from the North Pacific Subtropical Gyre.