Largely responsible for the oxygen in our atmosphere, cyanobacteria have been a dominant mechanism for converting—or fixing—carbon from carbon dioxide, making life as we know it possible. Today, these microorganisms are still important to the global carbon cycle because, in addition to fixing carbon, they also fix large quantities of nitrogen. Yet, we still do not fully understand how these organisms perform this critical function of nitrogen fixation. In collaboration with the University of Southern California, we are studying nitrogen fixation and photosynthesis in the marine cyanobacteria that play a critical role in global carbon cycling. By enhancing our understanding of microbial metabolism and the roles of individual microbial species, ecosystem researchers will be able to improve predictions of how these processes respond to and affect climate change.

Our approach is to expose the bacteria to stable isotope tracers and then map the tracer distribution in individual bacterial cells with the nanoscale secondary ion mass spectrometer (NanoSIMS)—an ultrahigh-resolution ion microprobe. Using cells labeled with stable isotopes of carbon and/or nitrogen, NanoSIMS imaging reveals locations of active growth and allows us to follow nutrient fluxes between cells.

Relevance to PLS Research Themes

NanoSIMS image of Anabaena
Figure 1. Using NanoSIMS, we have analyzed a chain of seven cells from a filament of Anabaena after four hours of incubation with carbon and nitrogen isotopic tracers. The secondary electron image (a) identifies the vegetative cells and specialized nitrogen-fixing cells, known as heterocysts. The enrichment of carbon (b) and nitrogen (c) is much lower in the heterocyst, confirming that the heterocyst is not fixing new carbon and is rapidly disbursing newly fixed nitrogen to neighboring cells.

Chemical imaging of biological materials is advancing our understanding of biochemical processes and the role of microbes in energy production, environmental remediation, and carbon sequestration. With the NanoSIMS, we aim to link microbial metabolism to molecular structures and produce a detailed view of how isotopically marked species propagate throughout individual cells.

These research areas are central to Livermore's role in the Genomics: GTL (previously known as Genomes-to-Life) program for the Department of Energy, which seeks to provide a system-level understanding of microbial processes essential to carbon cycling and the cycling of such other elements as nitrogen, phosphorous, sulfur, oxygen, and metals.

Furthermore, our efforts to quantitatively measure biochemical systems at subcellular scales have direct benefits in the forensic characterization of biological weapons materials. Major Accomplishments in 2005 One particular cyanobacteria, Trichodesmium (sea sawdust), may account for up to half of the N2 fixation in the North Atlantic Ocean. However, it is unclear how Trichodesmium simultaneously fixes both carbon and nitrogen because nitrogenase enzymes used in N2 fixation are strongly inhibited by the O2 produced during photosynthesis. Thus, these two critical processes must be either spatially or temporally isolated. Another genus of cyanobacteria, Anabaena, solves the incompatibility problem by isolating nitrogen fixation in specialized cells called heterocysts (Figure 1a). While these specialized cells are easily identified in Anabaena, specialized nitrogen-fixing cells have been suspected but never identified in Trichodesmium.

Combining transmission electron microscopy and NanoSIMS, we tracked uptake and redistribution of nitrogen and carbon at the cellular and subcellular level. We identified the exact sites where newly fixed carbon and nitrogen were allocated in cells grown in a 13C- and 15N-enriched environment. In the model organism Anabaena, newly fixed carbon is significantly lower in nitrogen-fixing heterocyst cells (Figure 1b), while neighboring vegetative cells are dramatically enriched because of their photosynthetic activity. Corresponding images indicate that, after being fixed in heterocysts, nitrogen is rapidly allocated to locations of new biomass growth in neighboring cell walls (Figure 1c).

In contrast, in experiments involving Trichodesmium, our analyses indicated few cell-to-cell differences in concentrations of newly fixed carbon, nitrogen, or phosphorus (Figure 2). A large portion of this newly fixed nitrogen was immediately confined to localized subcellular regions. These regions, called cyanophycin granules, are thought to be storage structures and temporary holding sites for nitrogen. In samples collected after 24 hours, these regions were less distinct and overall cell enrichment had risen dramatically. In conclusion, unlike Anabaena, which uses specialized nitrogen-fixing cells, Trichodesmium uses temporary nitrogen storage at the subcellular level as a primary mechanism of metabolic segregation. It also processes carbon and nitrogen at different times in the same cells.

Images of Trichodesmium
Figure 2. Unlike Anabaena, Trichodesmium does not isolate nitrogen fixation in specialized cells. Instead, it uses subcellular storage as indicated in these corresponding NanoSIMS and transmission electron microscope images. The micrograph (a) shows two Trichodesmium cells after eight hours of incubation with carbon and nitrogen. In images showing (b) carbon isotope, (c) nitrogen isotope, and (d) phosphorus-to-12C ratios, respectively, the cyanophycin granules, which are thought to store nitrogen, are clearly visible and are indicated by arrows.

Scientific Impact

Marine microbiologists have long sought to explain the paradoxical behavior of Trichodesmium;mdash;a nonheterocystous cyanobacteria that is able to fix both CO2 and N2 concurrently during the day. Several competing hypotheses have been proposed that support either temporal and spatial segregation mechanisms; however, our analyses with the NanoSIMS presented the first direct imaging of nitrogen and carbon behavior at the subcellular level. In general, these results support the temporal segregation model for Trichodesmium.

Lawrence Livermore National Laboratory Our experiments clearly illustrate how the NanoSIMS and stable isotope tracers can be used to track nutrient allocation at the individual cell and subcellular levels. This capability can be used to study a wide range of topics in the emerging fields of microbial proteomics and metabolomics, allowing us to shed light on critical functions of environmental microbes. This knowledge will be critical to modeling and bioengineering efforts in topics ranging from environmental cleanup to carbon sequestration and energy generation.

Related Publications

C. G. Marxer et al., "Supported Membrane Composition Analysis by Secondary Ion Mass Spectrometry with High Lateral Resolution," Biophys. J. 88, 2965-2975 (2005).

Contact: Jennifer Pett-Ridge [bio], 925-424-2882,

Jennifer Pett-Ridge

Piecing Together the Cyanobacteria Puzzle

New Frontiers

Several nitrogen- and carbon-fixing marine cyanobacteria genomes have recently been fully sequenced, providing unprecedented opportunities to link genes, proteins, and metabolic activities. Using NanoSIMS, we will study these organisms and genetically modified strains with one or more genes that have been made inoperative in critical functional processes. Once we understand the gene encoding for specific proteins, we will study the spatial location of these proteins and their movements within the cell during critical functions such as DNA replication, mitosis, and biomass growth. By linking the power of NanoSIMS and molecular genomic approaches, we hope to increase the speed with which we can discern microbial metabolic strategies in uncultured and novel organisms.