Periodic Reporting for period 4 - whyBOTher (Why does Clostridium botulinum kill? – In search for botulinum neurotoxin regulators) Reporting period: 2021-07-01 to 2022-12-31 Summary of the context and overall objectives of the project Bacterial toxins cause devastating diseases in humans and animals, ranging from necrotic enteritis to gas gangrene and tetraplegia. While toxin synthesis probably offers bacteria a selective advantage in competitive or hostile settings, toxigenesis is likely to represent a fitness cost to the bacterium. Therefore, bacterial populations probably adjust toxin production to competing metabolic requirements, and non-competitive environments select for phenotypes with silenced toxin production machinery. Understanding the genetic and cellular strategies bacteria use to attenuate toxin production in response to their environment is a key to interventions for disease prevention, antitoxin and vaccine development, and food and feed preservation. The overall objective is to understand the process of bacterial toxin production in its entirety, including the environmental and nutritional triggers that provoke or discourage bacteria to toxin production; how bacterial cultures coordinate toxin production and related or coupled traits; which genetic, regulatory and metabolic events direct the synthesis of toxins; how toxins are synthesized and transported; and eventually, why toxins are produced. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The work has focused on validation and refining research materials and methods, and on elucidating heterogeneity in bacterial cell populations and identifying novel regulators of toxin production. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Research on bacterial toxigenesis is mostly based on population-level net measurements of bacterial growth, gene expression, and toxin synthesis. These measurements are noisy and unstable, and mostly allow the study of all-on and all-off traits, whereas subtle links between weak traits get often missed. Single-cell biology provides a higher level of resolution to research on bacterial toxigenesis and allows detection of toxin gene activity or localization of toxin, and related traits, in individual cells. This allows matching of multiple traits and identification of variable trait patterns or weak links between traits. This gives hints to novel regulatory mechanisms, which is turn is a key to understanding cellular processes and, eventually, controlling toxigenesis.