Single-cell RNA sequencing of multicellular communities

• Applicant: Professorin Dr. Susanne Häußler
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 503910970

Multicellular bacterial communities exhibit various forms of heterogeneity, including co-occurrence of cells with different morphological traits, biochemical properties, and gene expression profiles. Furthermore, in biofilm-associated infections, genetic diversity of the infecting strain is a typical feature. This heterogeneity has been associated with higher productivity and lower susceptibility of the bacterial community to environmental changes including antibiotic treatment as well as altered virulence traits. Due to the need and importance of a better understanding of this heterogeneity, methods using e.g. fluorescent reporters or probes to examine a specific gene of interest, have been extensively applied. More recently, single-cell RNA sequencing (scRNA-seq) has become a powerful tool to examine heterogeneity of eukaryotic cells. This advanced technology is only at the beginning of its application for bacteria, as there were still some hurdles to overcome. The bacterial cell wall is incomparably more resistant to cell lysis, the mRNA has a shorter half-life and a much lower copy number, and isolating single cells from a multicellular community is a challenge. Nevertheless, first recent work has provided valuable insight into factors influencing microbial heterogeneity. In this proposal, we will establish single cell sequencing based on published protocols and study, how the microenvironment and genetic variability of Pseudomonas aeruginosa biofilm population shapes subpopulations and drives antibiotic tolerant phenotypes. We furthermore aim to contribute to the establishment of a SPP technology pipeline that involves single cell transcriptomics and FACS-based cell sorting. The integration of the data promises a detailed characterization of bacterial subpopulations of three model species (P. aeruginosa, Bacillus subtilis and Escherichia coli).

• DFG Programme: Priority Programmes
• Subproject of: SPP 2389: Emergent Functions of Bacterial Multicellularity