Final Report Summary - PHENOFIX (Biological Function and Evolution of Phenotypic Noise in N2-fixation on the Single-cell Level) Overview: Genetically identical cells that live in a homogeneous environment often show substantial variation in their biological traits; such variation is called phenotypic heterogeneity. The level of phenotypic heterogeneity has a genetic basis, suggesting that higher levels of phenotypic heterogeneity can evolve. In fact, recent theoretical studies suggest that phenotypic heterogeneity could be a mechanism for a population of genotypes to respond to uncertain environments in a more efficient way than with conventional signal transduction pathways. However, it is not known if phenotypic heterogeneity is relevant for bacterially-driven processes in the environment, because the few studies that experimentally investigated phenotypic heterogeneity in bacteria did not consider metabolic activities that contribute to biogeochemical cycles. While it has been observed with novel nanoSIMS (nano-scale secondary ion mass spectrometry) technology that bacteria display phenotypic heterogeneity in metabolic activities, direct experimental evidence that phenotypic heterogeneity in metabolic activities has a biological function and can provide isogenic bacterial populations with a growth advantage is missing on a single-cell level. Moreover, it has not been experimentally tested if phenotypic heterogeneity in metabolic activities adapts over evolutionary timescales in response to fluctuating environmental conditions. The goal of this project is to experimentally investigate how phenotypic heterogeneity affects bacterial metabolic activity, growth and evolution under fluctuating environmental conditions. The proposal focuses on phenotypic heterogeneity in N2-fixation in the unicellular aquatic bacterium Klebsiella pneumoniae. The experiments will combine time-lapse microscopy, nanoSIMS and experimental evolution to understand why bacteria display phenotypic heterogeneity in metabolic activities. This will establish a link between the behaviour of single cells and biogeochemical cycles, and reveal how variation at the single-cell level can impact processes at the ecosystem level.The objective was to answer these specific questions:1. What are the conditions under which genotypically identical N2-fixing K. pneumoniae establish subpopulations of actively N2-fixing cells in the presence of NH4+ due to phenotypic heterogeneity. 2. Do individual cells that perform N2-fixation in the presence of NH4+ have a growth advantage as compared to non-active cells after a sudden transition from replete to deplete NH4+.3. Do the phenotypic switching rate evolve in response to the frequency of NH4+ fluctuations. Work performed: We experimentally answered question (1) and (2). To this end, we developed new methodologies including single-molecule mRNA fluorescence in situ hybridization (smFISH) for Klebsiella nitrogenase and a nanoSIMS-based pulse-chase/pulse approach. Question (3) was not approached because a publication by another research group in the field during the course of the project showed in principle that the switching rate in a metabolic activity can evolve in response to environmental fluctuations (New et al., 2014, Plos Biology). Instead, we experimentally investigated the mechanism the establishes phenotypic heterogeneity in N2-fixation using correlative microscopy between nanoSIMS and smFISH. In addition, we initiated a project investigating the environmental relevance of phenotypic heterogeneity in metabolism in natural, non-laboratory environments.Main results: We performed experiments delineating the conditions under which K. pneumoniae shows phenotypic heterogeneity in nitrogen fixation. We find that phenotypic heterogeneity in nitrogen fixation emerges under limiting ammonium supply in chemostat environments. We show heterogeneity at the level of gene expression using smFISH and directly at the level of activity with nanoSIMS combined with 15N stable isotope labeling. We find that gene expression and activity do not correlate in single cells suggesting that cells switch between highly active and less-active states. We introduce a pulse-chase/pulse approach coupled to nanoSIMS that allows measuring the direct benefit of heterogeneity in any assimilatory metabolic activity in fluctuating environments. Using this approach we show that the individual investment in fixation of 15N2 during limited NH4+ supply is directly correlated to the growth rate after a switch to an NH4-deplete environment as determined by the uptake of 13C-glucose. Expected final results:Our data provide direct experimental evidence for predictions made by theoretical studies, which found that phenotypic heterogeneity in isogenic microbial populations can optimize growth in fluctuating environments. Importantly, the pulse-chase/pulse approach can now be used to study the adaptive benefit of heterogeneity in complex microbial communities living in natural, nutrient-limited environments, because it is independent of fluorescent protein-based heterogeneity measurements that are only possible in genetically tractable model organism grown in the laboratory. Socio-economic impact: The project shows that nutrient-limitation fosters bacteria to establish functionally relevant biodiversity at the level below genetic differences; i.e. the phenotype. This is important information for controlling bio-processes and understanding ecosystem functioning in general. The project promotes the understanding of the biology of N2-fixation, a key process in agriculture.