Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH, a transcriptome-imaging approach that records gene-expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We apply this approach to the opportunistic pathogen, Pseudomonas aeruginosa, analyzing ~600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identify numerous metabolic and virulence-related transcriptional states that emerge dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm, just several microns away, underscoring the importance of microenvironments. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.