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Chapter 15 : FISH-Microautoradiography and Isotope Arrays for Monitoring the Ecophysiology of Microbes Within Their Natural Environment
Category: Environmental Microbiology; Applied and Industrial Microbiology
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The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR), as well as the isotope array, which are described in this chapter, are both based on the use of radioactive isotopes for revealing a specific ecophysiology and rRNA for identifying the respective organisms and provide the direct means to identify microbes that catalyze a defined process in an ecosystem. In the combination of both approaches, DNA-stable isotope probing (SIP) serves to rapidly narrow down the number of 16S rRNA genes, which might originate from substrate-consuming microbes, and FISH-MAR is then used to prove the actual functional involvement of these bacteria. Isotope arrays benefit from the fact that rRNA is labeled faster than DNA after exposure of a cell to a suitable labeled substrate, but are less sensitive than FISH-MAR because a single biomarker and not all cellular compounds contribute to the radioactive signal. If used with 14CO2 as the activity marker, isotope arrays are able to detect metabolic activity of community members that comprise not more than 1% of all cells in the investigated sample. NanoSIMS-based techniques are even more sensitive than FISH-MAR and offer a reliable quantification of the incorporated radiotracer per cell, but do not provide information about the labeled compound classes in the cell, are much more timeconsuming, and can only be performed in a few laboratories worldwide.
FISH-MAR analyses of a novel member of the Betaproteobacteria in an ammonia-oxidizing enrichment culture. The left column displays the fluorescent signals after hybridization of the enrichment with a specific probe for the betaproteobacterium. The middle column shows the corresponding MAR signals, and the right column presents an overlay of both figures. (Top row) Dead control. The cells in the enrichment were killed prior to incubation with 14CO2 and 1 mM NH4 + for 16 h. No incorporation of 14CO2 is detectable, excluding chemography or labeling of the cells by the radiotracer in the absence of metabolic activity. (Middle row) The enrichment was incubated with 0.1 mM ammonium in the presence of 14CO2 for 16 h. The cells of the betaproteobacterium are MAR positive, indicating that the bacterium is an autotrophic ammonia oxidizer. (Bottom row) Same experiment as in the middle panel but in the presence of 1 mM ammonium. The cells of the betaproteobacterium are strongly labeled, demonstrating that this organism fixes more CO2 in the presence of 1 mM than in the presence of 0.1 mM. Scale bar corresponds to 10 μM. It should be noted that other microbes than the betaproteobacterium were present in the sample. These microbes are not visible in the FISH pictures, but they could have caused silver grain formation at spots where no cells of the betaproteobacterium were located. Photos by Roland Hatzenpichler.