Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, 00014 Helsinki, Finland.
Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, 00014 Helsinki, Finland.
Int J Food Microbiol. 2025 Jan 2;426:110927. doi: 10.1016/j.ijfoodmicro.2024.110927. Epub 2024 Oct 1.
Laboratory domestication is the result of genetic and physiological changes of organisms acquired during numerous passages in vitro. This phenomenon has been observed in bacteria as well as in higher organisms. In an effort to understand the impact of laboratory domestication on the foodborne pathogen Clostridium botulinum and related microbial food safety research, we investigated multiple spore stocks of C. botulinum Group II Beluga from our collection, as that is a widely applied model strain used in laboratories over decades. An acquired nutrient auxotrophy was confirmed as thymidine dependency using phenotypic microarrays. In parallel, whole-genome re-sequencing of all stocks revealed a mutation in thyA encoding thymidylate synthase essential for de-novo synthesis of dTMP from dUMP in the auxotrophic stocks. A thyA-deficient Beluga variant stock was successfully complemented by introducing an intact variant of thyA and thymidine prototrophy was restored, indicating that the thymidine auxotrophy was solely due to the presence of a SNP in thyA. Our data suggested that this mutation, deleterious under nutrient-poor growth conditions in a chemically defined medium, has been present and maintained in laboratory stocks for nearly 30 years. Yet, the mutation remained unidentified since receiving the strain, most likely due to routine use of culture conditions optimized for growth performance. This work pinpoints the need for careful monitoring of model strains extensively used in laboratory settings at both phenotypic and genomic level. In applications like food safety challenge tests, compromised strains could cause incorrect predictions and thereby have deleterious consequences. To mitigate the risk of acquiring mutations, we recommend keeping passage numbers of laboratory strains low and to avoid single-colony passaging. In addition, relevant strains should be subjected to regular WGS checks and physiological validation to exclude DNA mutations with potential negative impacts on research data integrity and reproducibility.
实验室驯化是生物在多次体外传代过程中获得的遗传和生理变化的结果。这种现象不仅在细菌中，而且在高等生物中都有观察到。为了了解实验室驯化对食源性致病菌肉毒梭菌及其相关微生物食品安全研究的影响，我们研究了我们收集的来自 II 组 Beluga 的多个孢子株，因为这是几十年来实验室中广泛应用的模型菌株。使用表型微阵列证实了获得的营养缺陷型是胸苷依赖性。同时，对所有菌株进行全基因组重测序发现，在依赖胸苷的菌株中，编码胸苷酸合酶的 thyA 发生突变，该酶对于从头从 dUMP 合成 dTMP 是必需的。通过引入完整的 thyA 变体，成功地对 thyA 缺陷型 Beluga 变体菌株进行了互补，恢复了胸苷原养型，表明胸苷缺陷型仅归因于 thyA 中的 SNP 的存在。我们的数据表明，这种突变在营养贫瘠的化学定义培养基中对生长不利，在实验室菌株中已经存在并维持了近 30 年。然而，由于常规使用优化生长性能的培养条件，该突变仍然未被识别。这项工作指出需要在表型和基因组水平上对实验室中广泛使用的模型菌株进行仔细监测。在食品安全挑战测试等应用中，受损菌株可能会导致错误预测，从而产生有害后果。为了降低获得突变的风险，我们建议将实验室菌株的传代次数保持在较低水平，并避免单菌落传代。此外，应定期对相关菌株进行 WGS 检查和生理验证，以排除可能对研究数据完整性和可重复性产生负面影响的 DNA 突变。
