Assessing the potential of mRNA-FISH FACS for isolation of functional soil bacterial populations for quantifying biogeochemical cycle interactions

This project ran for 9 months in 2012. The investigators were:

  • Principal investigator: Dr Liz Shaw, University of Reading, Department of Geography and Environmental Sciences
  • Co-Investigator: Professor Elizabeth Baggs, University of Aberdeen, School of Biological Sciences
  • Researcher-Co-Investigator: Dr Christos Gougoulias, now Syngenta Ltd


Our overall aim is to assess the potential of combining C isotope pulse chase with mRNA-Fluorescence in situ hybridisation (FISH) and flow cytometry-fluorescence-activated cell sorting (FC-FACS) to quantify interactions between assimilatory and dissimilatory biogeochemical processes within the soil microbial biomass.

For proof of concept, we will focus on the expression of nosZ (encoding nitrous oxide reductase; the final enzyme in the denitrification pathway) and our specific objectives are to:

1. Optimise mRNA-FISH for flow cytometry analysis of nosZ expressing soil-extracted bacterial cells

2. Establish and characterize denitrifying soil microcosms with respect to 14C-substrate pulse mineralization kinetics and relate this substrate utilisation to denitrifier 15N-N2O/N2 production

3. Combine 14C-substrate pulsing with mRNA-FISH and FC-FACS to test, as a case study, the hypothesis that the competitiveness of nosZ expressing bacteria for C will depend on the carbon source and be represented by a lowered denitrification 15N-(N2O-to-N2) product ratio.


The normal growth of all living entities depends on an adequate source of essential elements (e.g. C, N, S, P) and, in this respect, the Earth can be considered a closed system with the supply of essential elements being finite. Therefore, the recycling of these elements through the environment is fundamental to avoid exhaustion and microbes can be viewed as the 'engine room' that drive the component processes responsible for the recycling of these elements in the Earth's biogeochemical cycles. In cycling carbon, soil microbes utilise different organic and inorganic forms of carbon as energy and carbon sources resulting in the transfer carbon between environmental compartments. However, the carbon cycle does not operate on its own but it is closely metabolically linked with that of other essential elements either via the use of these as reductants and oxidants in energy transduction or via their incorporation into biomass (or release from decaying dead biomass) as part of multiple essential element- containing biomolecules (e.g proteins, DNA). Hence, the availability of carbon is a key factor in determining the transformations and cycling of other essential elements whilst the availability of other key elements control the rate at which microbes consume and respire carbon. Such biogeochemical cycle interactions can be illustrated by the soil microbial process of denitrification: the decomposition of organic carbon under low oxygen conditions through the respiration of nitrate resulting in the step-wise reduction of nitrate to dinitrogen gas (N2) with nitrous oxide (N2O) produced as an intermediate.

A central goal in microbial ecology is to link biogeochemical processes to specific microbial taxa in the environment so that the role of microbial community structure can be better represented in predictive models. A suite of methods have been developed in the last decade in order achieve this goal without the need for cultivation and characterization of isolates but none of these offer the opportunity to quantify the interactions between biogeochemical cycles in a microbially-oriented way, for example, with respect to the use of a particular carbon source as a reductant to drive denitrification. Gaining the quantitative understanding of the interactions that is required to predict essential element fluxes and feedbacks under perturbed carbon cycle and environmental change scenarios is therefore method- limited.

This project will provide proof-of-concept of a new method to quantify use of carbon by bacteria whilst transforming another essential element. The bacterial denitrification pathway will serve as a case study with a focus on the bacteria using carbon to reduce N2O to N2 (the final step in denitrification) due to the crucial role that this group play in regulating the atmospheric concentration of N2O, a potent greenhouse gas. The new method involves: (i) use of C isotopes to trace microbial C consumption; (ii) labelling actively N2O-reducing microbial cells with a fluorescent dye; (iii) sorting the fluorescent cells and quantifying the C isotope content.

The proof of concept will be in simple experimental systems involving known N2O-reducing bacteria and soil microcosms incubated under conditions known to promote denitrification. As a case study, we will test a theory concerning the carbon source preference of the N2O-reducing bacteria. The project brings together the complimentary expertise of the investigators (use of C isotopes, fluorescence-labelling and sorting of bacteria, denitrification biogeochemistry) and the project partner (fluorescence labelling of bacteria active in biogeochemical cycling). We will use state-of-the art stable isotope techniques to quantify microbial N2O reduction and exploit advances in instrumentation for cell sorting that enables the accurate detection of bacterial cells extracted from soil.

Methodology overview

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