Quantitative Imaging of Nitrogen Fixation by Individual Bacteria Within Animal Cells

Abstract

Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonia for biosynthesis, is exclusively performed by a few bacteria and archaea. Despite the essential importance of biological nitrogen fixation, it has been impossible to quantify the incorporation of nitrogen by individual bacteria or to map the fate of fixed nitrogen in host cells. In this study, with multi-isotope imaging mass spectrometry we directly imaged and measured nitrogen fixation by individual bacteria within eukaryotic host cells and demonstrated that fixed nitrogen is used for host metabolism. This approach introduces a powerful way to study microbes and global nutrient cycles.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

“…Because ion count rates can vary with instrument tuning, sample topography, and other concentration-independent factors, quantitative 13 C-and 15 N-enrichment images were constructed by normalizing the lipidspecific ion counts at each pixel to the major species counts (i.e., 13 C 1 H − / 12 C 1 H − or 12 C 15 N − / 12 C 14 N − ). This normalization works because the concentration-independent variations in count rates for the lipid-specific rare isotope (i.e., 12 C 15 N − ) are proportional to the concentration-independent variations in count rates for the corresponding abundant isotope species (i.e., 12 C 14 N − ) (14,24,25), as demonstrated by control experiments (Fig. S2).…”

“…The resulting ratios are proportional to the local abundance of the isotopically labeled molecules. These ratios were divided by standard natural abundance ratios, producing "isotope enrichment factors" that quantify the amount of 13 Clipids and 15 N-sphingolipids compared with unlabeled cells (24) (Fig. S2).…”

Symbiosis as a General Principle in Eukaryotic Evolution

“…The access to atmospheric nitrogen in these plants facilitates their utilization of nitrogenpoor soils, for example, in early succession communities, and is recognized as a contributory factor to the evolutionary diversification of legumes, with their large, nitrogen-rich seeds protected by nitrogen-containing toxins, including alkaloids (Houlton et al 2008;Corby et al 2011). Nitrogen-fixing symbioses have also been reported in protists, for example, in marine diatoms (Foster et al 2011), and some animals, notably the wood-feeding shipworms and some termites (Lechene et al 2007;Desai and Brune 2012).…”

A single-cell view of ammonium assimilation in coral–dinoflagellate symbiosis

“…Metabolic studies on coral-dinoflagellate symbioses generally involve the unnatural culture of both symbiotic partners separately or the separation of algal and host tissue fractions, an experimental procedure, which is often incomplete and hampered by crosscontamination (Yellowlees et al, 2008). In this context, recent advances in a high-resolution isotopic imaging technology, the sub-micrometer scale ion microprobe technique referred to as NanoSIMS provides direct imaging and quantification of the metabolic exchanges within an intact symbiosis following stable isotope labeling (Lechene et al, 2006(Lechene et al, , 2007Kuypers, 2007;Musat et al, 2008;Dattagupta et al, 2009;Foster et al, 2011). Nano-SIMS has recently been successfully applied to reefbuilding corals in order to image trace-elemental distributions in different ultra-structural components of both fossil and living corals (Meibom et al, 2004(Meibom et al, , 2008Clode et al, 2007;Reynaud et al, 2007;Stolarski et al, 2007;Houlbreque et al, 2009;Brahmi et al, 2010).…”