Exploring the morphological heterogeneity of \(Bacteroides\) \(thetaiotaomicron\) using a low-input RNA-sequencing protocol

Genetically identical bacteria can be phenotypically diverse. This phenotypic heterogeneity arises from a range of cellular processes and equips bacterial communities with a versatile toolkit to withstand environmental stress, exploit new niches, and rapidly adapt to fluctuating conditions, ultimately enhancing their survival. This is exemplified by the abundant gut microbiota species Bacteroides thetaiotaomicron. Constantly exposed to its host and other members of the microbiota, Bacteroides thetaiotaomicron is a very heterogeneous species. It provides colonization resistance against enteric infection in a healthy gut environment, and is able to drastically change its role in a disbalanced microbiota by cross-feeding pathogens. In addition to functional heterogeneity, B. thetaiotaomicron is morphologically heterogeneous. Its name reflects the three distinct morphologies (dubbed ‘Theta’, ‘Iota’ and ‘Omicron’) that these bacteria can adopt. However, the molecular basis of morphological heterogeneity in Bacteroides and potential functional consequences thereof are unknown. In this thesis, B. thetaiotaomicron morphotypes are systematically characterize during growth in laboratory medium and within host-associated niches. To distinguish these morphotypes, a fluorescence-activated cell sorting (FACS)-based strategy is employed, enabling their separation based on size. Subsequently, a sensitive, low-input transcriptomic approach is used to profile the gene expression signatures associated with each defined morphotype. The derived candidate morphological marker genes, encoding proteins involved in primary metabolism and membrane- associated processes, are validated using single-molecule fluorescence in-situ hybridization. Causal gene-phenotype links are inferred from single-cell imaging of individual members of a transposon library and corroborated by analyzing clean deletion and overexpression mutants. To investigate additional layers of heterogeneity within a morphologically defined population of cells, single-bacteria RNA-seq was applied and revealed a distinct supplementary level of heterogeneity. Overall, this work lays the ground to dissect the extent and impact of cell-to-cell variability in this dominant species of the human microbiota.