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 N(2) and CO(2) fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with (15)N(2) and (13)CO(2) under in situ conditions with and without NH(4)(+). 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 N(2) in the absence of NH(4)(+), but not in the presence of NH(4)(+) as has previously been suggested. N(2) and CO(2) fixation were heterogeneous among cells and positively correlated indicating that N(2) and CO(2) 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.