HR: 10:50h
AN: B52A-03    [Abstracts]
TI: Visualizing Single Cell Biology: Nanosims Studies of Carbon and Nitrogen Metabolism in Diazotrophic Cyanobacteria
AU: * Pett-Ridge, J
EM: pettridge2@llnl.gov
AF: Lawrence Livermore National Lab, Chemical Sciences Division, P.O. Box 808, L-231, Livermore, CA 94551, United States
AU: Finzi, J A
EM: finzi@usc.edu
AF: University of Southern California, Wrigley Institute for Environmental Studies & Department of Biological Sciences, 3616 Trousdale Parkway, AHF 108, Los Angeles, CA 90089, United States
AU: Capone, D G
EM: capone@usc.edu
AF: University of Southern California, Wrigley Institute for Environmental Studies & Department of Biological Sciences, 3616 Trousdale Parkway, AHF 108, Los Angeles, CA 90089, United States
AU: Popa, R
EM: rpopa@pdx.edu
AF: Portland State University, Department of Biology, P.O. Box 751, Portland, OR 97207, United States
AU: Nealson, K H
EM: KNealson@jcvi.org
AF: University of Southern California, Wrigley Institute for Environmental Studies & Department of Biological Sciences, 3616 Trousdale Parkway, AHF 108, Los Angeles, CA 90089, United States
AU: Ng, W
EM: wng@stanford.edu
AF: Stanford Univeristy, 318 Campus Drive, Clark Center E-250, Stanford, CA 94305, United States
AU: Spormann, A M
EM: spormann@stanford.edu
AF: Stanford Univeristy, 318 Campus Drive, Clark Center E-250, Stanford, CA 94305, United States
AU: Hutcheon, I D
EM: hutcheon1@llnl.gov
AF: Lawrence Livermore National Lab, Chemical Sciences Division, P.O. Box 808, L-231, Livermore, CA 94551, United States
AU: Weber, P K
EM: weber21@llnl.gov
AF: Lawrence Livermore National Lab, Chemical Sciences Division, P.O. Box 808, L-231, Livermore, CA 94551, United States
AB: Filamentous nitrogen fixing (diazotrophic) cyanobacteria are key players in global nutrient cycling, but the relationship between CO₂- and N₂-fixation and intercellular exchange of these elements remains poorly understood in many genera. These bacteria are faced with the challenge of isolating regions of N-fixation (O₂ inhibited) and photosynthetic (O₂ producing) activity. We used isotope labeling in conjunction with a high-resolution isotope and elemental mapping technique (NanoSIMS) to quantitatively describe ¹³C and ¹⁵N uptake and transport in two aquatic cyanobacteria grown on NaH¹³CO₃ and ¹⁵N₂. The technical challenges of tracing isotopes within individual bacteria can be overcome with high resolution Secondary Ion Mass Spectrometry (NanoSIMS). In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs⁺, O^-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio ‘map' can then be generated for the analyzed area. Using sequentially harvested cyanobacteria in conjunction with enriched H¹³CO₃ and ¹⁵N₂ incubations, we measured temporal enrichment patterns that evolve over the course of a day's growth and suggest tightly regulated changes in fixation kinetics. With a combination of TEM, SEM and NanoSIMS analyses, we also mapped the distribution of C, N and Mo (a critical nitrogenase co-factor) isotopes in intact cells. Our results suggest that NanoSIMS mapping of metal enzyme co-factors may be a powerful method of identifying physiological and morphological characteristics within individual bacterial cells, and could be used to provide a 3-dimensional context for more traditional analyses such as immunogold labeling. Finally, we resolved patterns of isotope enrichment at multiple spatial scales: sub-cellular variation, cell-cell differences along filaments, inter-species transfers (with Rhizobium epibionts), and within-cell depth profiles. Spatial enrichment patterns were correlated with morphological features evidenced in TEM images of microtomed filaments. These features indicate how 15N and 13C "hotspots" are dispersed throughout individual cells in different species, and may indicate isolated locations of increased N₂ fixation, sites of amino acid/protein synthesis, or cyanophycin storage granules. This combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis and high resolution microscopy allows isotopic analysis to be linked to morphological features and holds great promise for fine-scale studies of bacteria metabolism.
DE: 0414 Biogeochemical cycles, processes, and modeling (0412, 0793, 1615, 4805, 4912)
DE: 0428 Carbon cycling (4806)
DE: 0460 Marine systems (4800)
DE: 0463 Microbe/mineral interactions
DE: 0471 Oxidation/reduction reactions (4851)
SC: Biogeosciences [B]
MN: 2007 Fall Meeting