Deep single-cell phenotyping to identify governing principles and mechanisms of the subcellular organization of bacterial replication

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. Our goal 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 single-cell phenomics approach 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. In doing so, we hope to transform our understanding of bacterial cell biology by expanding it beyond current textbook standards and provide us with the blueprints and design principles of bacterial cells.

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Mutational robustness and evolvability in model microbes

Organisms need to balance the need for mutations to evolve with mitigating these mutations’ potential negative effects. One hypothesis is that some cellular mechanisms help minimize the phenotypic consequences of mutations and hence confer mutational robustness. However, whereas the concept of mutational robustness is central to our understanding of evolution and genetics, our knowledge about it remains surprisingly limited. This is because studying robustness is extremely challenging: it requires interrogating the role of a large number of genes on the effect of a large number of (de novo or standing) mutations on several traits and in several environments. We aim to combine the power of two model organisms, E. coli and S. cerevisiae, with an evolutionary modelling framework to identify genes and mechanisms associated with reducing mutational effects, assess the type of mutations that can be mitigated and model and test if and how mutational robustness influences evolvability.