PIs: Barbara J. MacGregor (University of North Carolina, Chapel Hill), Andreas P. Teske (UNC)
Amount: $49,457.29
Award Dates: January 1, 2013 — December 31, 2014

Abstract

Detection of bacterial and archaeal cells, and isolation and analysis of their DNA and RNA, is challenging where populations are sparse and activities low. In “click” chemistry, so far applied mainly in eukaryotes, target molecules incorporate a label that can be specifically tagged for capture or microscopic detection under gentle reaction conditions. Ideally the labeling, tagging, and capture or detection efficiencies are high, and the label does not affect growth or metabolism. We have been investigating whether bacteria can incorporate the “clickable” RNA analog 5-ethynyl uridine (EU) for subsequent attachment of fluorescent label (Alexa Fluor) for microscopic detection or a chemical tag (desthiobiotin) for streptavidin-mediated RNA capture. The results for both aspects so far are a qualified “yes”. Both Escherichia coli and Bacillus subtilis grown with EU can subsequently be detected by fluorescence microscopy, but E. coli becomes considerably more brightly labeled. It also exhibits a slight but reproducible growth inhibition, and a distribution of fluorescence intensity across the population that becomes increasingly uneven over a one-day incubation. This might be due to normal physiological variation, or to the slower growth and hence slower label dilution rate of labeled cells. If the latter, the implication for natural samples is that slowly growing cells might actually become more strongly labeled. For the desthiobiotin labeling, both pure culture and environmental RNA has been captured, as shown on gels, but sequencing has been unsuccessful. We expect that this is a solvable methodological problem, and should not discourage others from exploring these methods.

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