Abstract
In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific 13C uptake during labelled methanol anaerobic degradation.
Highlights
Over the past decades, molecular biology approaches have allowed to overcome limitations associated to cultivation-dependent methods, which were greatly underestimating actual diversity [1], and enabled strong advances in microbial communities’ quantification and classification
New methodologies involving high spatial resolution secondary ion mass spectrometry have been developed [6,7,8,9]. They allow more precise isotopic measurements and a better spatial resolution than previous techniques. These methods all rely on the combination of rRNA-based in situ hybridization with stable isotope imaging based on nanoSIMS, they differ notably by the hybridization procedure
The isotopic values are nearly identical for untreated cells, fixed cells and fixed/hybridized cells, which means that the procedures used for SIMSISH have a very limited influence on the apparent isotopic composition of microbes measured with elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) at population level
Summary
Molecular biology approaches have allowed to overcome limitations associated to cultivation-dependent methods, which were greatly underestimating actual diversity [1], and enabled strong advances in microbial communities’ quantification and classification. Linking phylogenetic information to function in complex environmental communities is one of the main challenges of microbial ecology In this context, different methods coupling isotope-labelling experiments and in situ hybridization have been developed to investigate the ecophysiology of microbial populations. Secondary Ion Mass Spectroscopy In situ Hybridization, SIMSISH, is based on the direct hybridization of halogenated DNA probes [6] whereas ELFISH or HISHSIMS (respectively Element Labeling-Fluorescent In situ Hybridization and Halogen In situ Hybridization Secondary Ion Mass Spectroscopy) uses catalyzed reporter deposition fluorescence in situ hybridization [10] with horseradish-peroxidase-labeled oligonucleotide probes and halogen-containing tyramides for the identification of microorganisms [7,8,9] These methodological differences could imply different consequences on the measurement of isotopic enrichment in cells. No detailed descriptions of these effects are available up to date
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
