A method for the use of the radiotracer 55Fe for microautoradiography and CARD-FISH of natural bacterial communities

Heterotrophic bacteria are key players in the biogeochemical cycle of iron (Fe) in the ocean, but the capability of different bacterial groups to access this micronutrient is ignored thus far. The aim of our study was to develop a protocol for the combined application of microautoradiography (MICRO) and catalyzed reporter deposition–fluorescence in situ hybridization (CARD-FISH) using the radioisotope ⁵⁵Fe. Among the different washing solutions tested, Ti-citrate-EDTA was the most efficient for the removal of extracellular ⁵⁵Fe providing sufficiently low background values. We further demonstrate that the washing of cells with Ti-citrate-EDTA and the fixation with paraformaldehyde or formaldehyde do not induce leakage of intracellular ⁵⁵Fe. Incubating natural bacterial communities collected from contrasting environments, the NW Mediterranean Sea and the Southern Ocean, with ⁵⁵Fe revealed that 3–29% of bacterial cells were associated with silver grains. Combining microautoradiography with CARD-FISH, we demonstrate that the contribution of different bacterial groups to total ⁵⁵Fe-incorporating cells was overall reflected by their relative contribution to abundance. An exception to this pattern was the proportionally higher contribution of Gammaproteobacteria, SAR86 and Alteromonas. Our study demonstrates the feasibility of MICRO-CARD-FISH using the radiotracer ⁵⁵Fe and provides the first description of marine bacterial assemblages actively incorporating Fe.