Unraveling the phenotypic landscape of bacteria using single-cell transcriptome imaging

Individual bacteria within isogenic populations can exhibit different phenotypes due to variation in gene expression. Phenotypic heterogeneity and plasticity can provide many advantages to a population, including robustness to rapid environmental changes such as exposure to antibiotics, and a natural mechanism for division-of-labor interactions. Despite its clinical and environmental importance, cell-cell variation in bacteria remains largely unexplored due to the technical challenges of systematically profiling single bacterial cells.

Using parallel and sequential fluorescence in situ hybridization (par-seqFISH), a transcriptome-imaging approach, we can now profile the expression of hundreds of genes at a single-cell and molecule resolution in diverse bacteria. However, achieving a global understanding of phenotypic variation requires the ability to measure the expression thousands of genes per experiment. Here, we are further developing our existing framework to produce "par-seqFISH 2.0", which will provide the technological basis for conducting transcriptome-scale studies of cell-cell variation across many conditions and bacterial species. Using this approach, we plan to explore the currently hidden phenotypic landscape of Pseudomonas bacteria both in lab cultures and in-vivo during infection. We are particularly interested in how single-cell heterogeneity manifests in plant-associated bacteria and opportunistic pathogens. By extending this method, we will be able to profile cell states at the transcriptome scale with single-molecule sensitivity across species and conditions.