Detection and Capturing of (14)C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads.

Abstract

Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO2 into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with (14)C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The (14)C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18Bqng(-1) detected after 25days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the (14)C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized.