PerspectiveMicrobiology

Sizing Up the Uncultivated Majority

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Science  14 Sep 2007:
Vol. 317, Issue 5844, pp. 1510-1511
DOI: 10.1126/science.1148538

Coupling the identity of microbes with their activity in the environment remains an important gap in our ability to explore microbial ecology. The development of techniques to quantify the metabolic activity of single microbial cells has been especially challenging, mostly due to their small size. Microbiologists are therefore excited about a new high-resolution imaging method called multi-isotope imaging mass spectrometry (MIMS) or nanoSIMS, which can help decipher what individual microbes are “doing” in the environment. On page 1563 of this issue (1), Lechene and colleagues apply MIMS to identify a symbiotic relationship between a nitrogen-fixing bacterium and an animal host. The technique is poised to reveal the metabolic diversity of the planet's microorganisms, 99% of which has eluded cultivation (2).

MIMS can determine the chemical, radioisotopic, and stable-isotopic composition of biological material down to the submicrometer level (3-6). By exposing microbial communities to substrates that have been labeled with stable isotopes, MIMS-based imaging allows visualization of metabolic activity in single cells. Moreover, nutrient uptake rates and fluxes can be quantified.

Lechene et al. used MIMS to quantify nitrogen (N2) fixation by individual bacteria that inhabit the gills of the shipworm Lyrodus pedicellatus. L. pedicellatus is a wood-eating marine bivalve with little nitrogen in its diet and must therefore rely on other nitrogen sources (7). Previous studies reported N2 fixation for intact shipworms, as well as for pure cultures of bacterial symbionts isolated from shipworm gills (7, 8), but neither the site of fixation nor whether the fixed nitrogen is supplied to the host could be determined. Lechene et al. grew shipworms in seawater containing nitrogen gas enriched in the rare stable isotope 15N and used MIMS to measure 15N incorporation in symbionts and shipworm tissue (see the figure). The incorporation of 15N was determined by comparing the quantitative mass images of 12C14Nand 12C15N—produced by bombardment of tissue with a cesium ion beam—to measure the increase in 15N/14N ratios relative to the natural abundance ratio (0.00367). Transmission electron microscopy of the same shipworm gill tissue was used to identify bacteria and host cells. The combined data provide the first direct evidence for in situ N2 fixation by bacterial symbionts and demonstrate that this nitrogen is used by the shipworm host.

Until the work of Lechene et al., it had not been possible to quantify the incorporation of nitrogen by individual N2-fixing microorganisms or to map the fate of fixed nitrogen in the microbial environment. Other methods currently used either do not provide single-cellresolution or, like micro-autoradiography, require that microorganisms be fed radioactive-labeled substrates (9). The uptake of radiolabeled isotopes directly links individual microbial cells to their activity in the environment. However, because this approach requires radioactivity, its use is limited to elements that have a radioisotope with a suitable half-life (>1 day; for example, 14C and 3H) and excludes the study of other elements such as nitrogen. MIMS, on the other hand, can be used to measure the distribution of any stable isotope as well as any radioisotope with a suitable half-life. Hence, the approach used by Lechene et al. holds great promise for studying symbiont-host interactions and microbial activity in the environment.

A new window on microbial activity.

The incorporation of 15N stable isotope into a mixed population of cells (animal cells and bacteria) is determined by comparing two quantitative mass images (12C14N and 12C15N) obtained by multi-isotope imaging mass spectrometry (MIMS). The increase in 15N/14N ratios relative to the natural abundance ratio can then be measured to identify the fate of the 15N.

PHOTO CREDIT: RUTH DIXON TURNER (DECEASED)/ALEXANDER AGASSIZ PROFESSOR AND CURATOR OF MALACOLOGY AT THE HARVARD UNIVERSITY MUSEUM OF COMPARATIVE ZOOLOGY

Combining MIMS with fluorescence in situ hybridization (FISH) is an even more powerful technique for identifying and characterizing single microbial cells. FISH uses fluorescent-labeled probes that are specific to the organism of interest and that bind to the intracellular 16S ribosomal RNA (2). Replacing fluorescent probes with isotopically labeled (stable or radioactive) or halogenated probes would allow individual cells to be directly identified (by probe hybridization to targets) by MIMS (10). The hybridization procedure is essentially identical to that used for FISH, and the same probes can be applied. By combining this probing technique with isotope labeling of substrate, one can assess the metabolic activity of cells and simultaneously identify their phylogenetic characteristics during a single MIMS scan. This approach links the identity of microbial cells to their in situ activity. MIMS is truly an imaging breakthrough, whose application is only just beginning to yield information once considered inaccessible.

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