Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS.
Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with (15)N2 and (13)CO2 under in situ conditions with and without NH4 (+). Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2% to now 26.5% of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary ion mass spectrometry to measure the incorporation (15)N and (13)C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4 (+), but not in the presence of NH4 (+) as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic heterogeneity in a rare bacterial species in its natural habitat, thus opening the door to study the occurrence and relevance of phenotypic heterogeneity in nature.
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Molecular Microbial Ecology Group, Department of Environmental Microbiology, Eawag - Swiss Federal Institute of Aquatic Science and Technology Zurich, Switzerland.
Matthias
M
Zimmermann
Department of Environmental Microbiology, Helmholtz-Centre for Environmental Research, Leipzig Germany.
Stéphane
S
Escrig
Department of Environmental Microbiology, Helmholtz-Centre for Environmental Research, Leipzig Germany.
Thomas
T
Hübschmann
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Department of Surface Waters, Eawag - Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum Switzerland.
Mathias K
MK
Kirf
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Department of Surface Waters, Eawag - Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum Switzerland.
Andreas
A
Brand
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Molecular Microbial Ecology Group, Department of Environmental Microbiology, Eawag - Swiss Federal Institute of Aquatic Science and Technology Zurich, Switzerland.
R Fredrik
RF
Inglis
Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research, Leipzig Germany.
Niculina
N
Musat
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland.
Susann
S
Müller
Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering, École Polytechnique Fédérale de Lausanne Lausanne, Switzerland ; Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, Lausanne Switzerland.
Anders
A
Meibom
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Molecular Microbial Ecology Group, Department of Environmental Microbiology, Eawag - Swiss Federal Institute of Aquatic Science and Technology Zurich, Switzerland.
Martin
M
Ackermann
Department of Environmental Systems Sciences, ETH Zurich - Swiss Federal Institute of Technology Zurich, Switzerland ; Molecular Microbial Ecology Group, Department of Environmental Microbiology, Eawag - Swiss Federal Institute of Aquatic Science and Technology Zurich, Switzerland.
Frank
F
Schreiber
FACS; Lago di Cadagno; dinitrogen fixation; diversity; green sulfur bacteria; phenotypic noise; phenotypic variability; single-cell analysis