Highly Multiplexed Spatial Transcriptomics in Bacteria.

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Bacterial single-cell behaviors are crucial but challenging to study due to high mRNA density.
• Existing imaging techniques struggle with the dense mRNA in bacteria, limiting transcriptomic analysis.

Purpose of the Study:

• To develop a high-throughput, spatially resolved method for profiling RNA in individual bacteria.
• To overcome the limitations of dense bacterial mRNA in image-based transcriptomics.

Main Methods:

• Combined 1000-fold volumetric expansion with multiplexed error-robust fluorescence in situ hybridization (MERFISH).
• Developed bacterial-MERFISH for high-throughput, spatially resolved RNA profiling in single bacteria.

Main Results:

• Successfully profiled thousands of operons within individual bacteria.
• Dissected *E. coli*'s response to carbon starvation.
• Mapped subcellular RNA organization and charted *B. thetaiotaomicron*'s adaptation to the gut environment.

Conclusions:

• Bacterial-MERFISH enables unprecedented insight into bacterial single-cell heterogeneity.
• The technique is broadly applicable to studying bacteria in diverse, native environments.
• This method opens new avenues for understanding bacterial adaptation and organization.