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Transcriptional profiles underlying morphological heterogeneity in Bacteroides thetaiotaomicron

ABSTRACT: Genetically identical bacteria can be phenotypically diverse. This is exemplified in the Bacteroidota, a predominant bacterial phylum of the human gut microbiota. However, the molecular basis of morphological heterogeneity in Bacteroidota species and potential functional consequences thereof are poorly understood. Here, we systematically characterize the cell morphology of Bacteroides thetaiotaomicron during its growth in laboratory culture and upon isolation from different host niches. We then develop a sensitive ‘mini-bulk’ transcriptomics approach to chart cell size-related gene expression patterns. The protocol builds on the MATQ-seq method that we here optimize for B. thetaiotaomicron, combined with CRISPR/Cas-based depletion of ribosomal sequences. We demonstrate that our transcriptomic pipeline accurately quantifies gene expression on a genomic scale. Its application to sort-collected B. thetaiotaomicron cells of different size enables us to derive morphological marker genes whose expression is enriched in bacterial cells of defined sizes. These morphological marker genes—encoding e.g. proteins involved in cell cycle, primary and secondary metabolism, and membrane-associated processes—are validated using single-molecule fluorescence in-situ hybridization. As microbial heterogeneity has become an emergent theme in microbiota research, this study may serve as a blueprint to link different phenotypes with functions in ever-increasing resolution.

ORGANISM(S): Bacteroides thetaiotaomicron

PROVIDER: GSE289312 | GEO | 2025/02/28

REPOSITORIES: GEO

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Project description:The adult human gut microbial community is typically dominated by two bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from E. rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the former possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole genome transcriptional profiling of both organisms in their distal gut (cecal) habitat as well as host responses, high resolution proteomic analysis of cecal contents, and biochemical assays of carbohydrate metabolism. B. thetaiotaomicron adapts to E. rectale by upregulating expression of a variety of polysaccharide utilization loci (PULs) encoding numerous glycoside hydrolase gene families, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is utilized by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of major gut bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability. The interactions between E. rectale and B. thetaiotaomicron were characterized by performing whole genome transcriptional profiling of each species after colonization of gnotobiotic mice with each organism alone, or in combination. E. rectale was also profiled during in vitro growth.

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Transcriptional profiles underlying morphological heterogeneity in Bacteroides thetaiotaomicron

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