Periodic Reporting for period 1 - BacterialBlueprint (Deep single-cell phenotyping to identify governing principles and mechanisms of the subcellular organization of bacterial replication)
Berichtszeitraum: 2022-09-01 bis 2025-02-28
Modern metagenomics has opened our eyes to the immense bacterial diversity that exists both among and within us. Despite this diversity, all bacteria share the basic challenge of organizing the various processes that ensure their faithful replication. All bacterial cells need to metabolize nutrients, generate building blocks, maintain their shape and size, replicate and segregate their chromosomes, synthesize cell walls and membranes, and divide to give rise to daughter cells. At present, we do not understand how bacteria integrate all these processes in their small cellular compartments. What makes this question even more intriguing is that bacteria represent simple forms of proliferating cells, without additional layers of internal organization (e.g. membrane-enclosed organelles) or cell cycle regulation (e.g. cyclins and cyclin-dependent kinases) seen in eukaryotic cells. The goal of the BacterialBlueprint project is to address this gap by uncovering the internal architecture of bacterial replication and identifying the molecular mechanisms that underlie it. For this, we rely on a high-throughput image-based profiling approach for bacterial cells that provides high-content, quantitative cell biological information. By applying this approach across different levels of bacterial diversity (both within and across species, beyond the small number of currently existing model species), we aim to identify general and species-specific principles for the subcellular organization of replication in bacteria. This analysis will also enable the identification of key factors involved in establishing these governing principles, which will be functionally characterized further to provide a unique overview of the molecular mechanisms that determine the spatial organization of bacterial replication. If successful, this project will transform our understanding of bacterial cell biology by expanding it beyond current textbook standards and providing us with the blueprints and design principles of bacterial cells.
Over the last two and a half years, we have assembled a large-scale collection of different bacterial species and strains. Our collection currently comprises >750 strains of >400 species, including >70 anaerobic gut microbiome members. We have developed a combinatorial staining protocol that enables visualization and quantification of cell biological and cell cycle properties across a diverse range of bacterial species. We are currently in the process of applying this staining protocol across our collection of bacteria. To achieve the required throughput for this project, we have improved our pipeline for image analysis, by implementing new methods for image segmentation (that are both more accurate and faster), by automating segmentation curation (see our recent STAR protocols publication) and by designing additional cellular features to be extracted from the cellular macromolecules that are visualized by the staining protocol. As a proof-of-concept, we applied part of this approach to Bacteroides thetaiotaomicron and Roseburia intestinalis, two prevalent human gut microbiome members. Comparison of both species revealed distinct cellular and cell cycle properties during exponential growth and upon nutrient depletion in stationary phase. We could also link the different cell biological response of both species to nutrient depletion to distinct alterations in gene expression. In addition, we have developed different transposon delivery vectors for use in anaerobic species, and are currently optimizing a conjugation and transposition protocol for constructing transposon mutant collections in gut microbiome members. In parallel, we used an existing Escherichia coli image-based profiling dataset to identify “cell cycle laws”, simple principles by which E. coli cells couple different cell cycle events to different aspects of cell size to achieve robust proliferation. It are these types of governing principles that we aim to identify across species and mutant libraries during the rest of this project. Using this E. coli dataset, we have also identified five genes of unknown function that lead to deviating cellular phenotypes upon their deletion. To assess their conservation, we have set up a bioinformatics pipeline, which we can readily adapt to other species in the future. We also further characterized these genes by examining protein structure and intracellular localization. We aim to expand such analyses to non-model species during the remainder of this project.
Our results so far constitute a major step forward in expanding bacterial cell biology beyond the intracellular organization of a limited number of well-established model species, which likely do not reflect the extensive diversity of the bacterial domain. One example of this is the unique peptidoglycan synthesis pattern we uncovered in R. intestinalis. As such, we believe our approach has the potential to enable future investigations of the cell biological diversity of many other gut microbiome members, and other non-model bacterial species in general. At the same time, the examination of shared processes across species enables direct comparison between them (as we did for B. thetaiotaomicron and R. intestinalis), but also the identification of common principles. The planned construction and imaging of mutant libraries in a diverse range of species will enable the identification and further characterization of the molecular mechanisms that underlie these governing principles. Together, this project will provide a comprehensive and quantitative understanding of the internal architecture of bacterial cells, thereby rewriting our textbook knowledge of bacterial cell biology.