Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, United States of America.
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States of America.
PLoS One. 2021 Mar 17;16(3):e0235303. doi: 10.1371/journal.pone.0235303. eCollection 2021.
Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.
连续培养系统允许在很长一段时间内控制微生物的生长。在这里，我们开发了一种新的诱变测试方法，该方法涉及在连续培养系统中培养酵母，使其暴露于低水平的诱变剂中约 20 天。相比之下，大多数基于微生物的诱变测试方法将潜在的诱变剂在短时间内以高浓度暴露于生物报告物。我们的测试方法将两种通过不同机制（嵌入剂溴化乙锭和烷化剂甲基甲磺酸酯）起作用的诱变剂的灵敏度提高了至少 20 倍，超过了成熟的艾姆斯测试。为了进行测试，培养物在含有（潜在）诱变剂的小型、廉价的连续培养系统中生长，通过两种方法评估添加化合物的致突变性：基于瓜氨酸的平板测定法和全基因组测序。在基于瓜氨酸的平板测定法中，我们能够在暴露于甲基甲磺酸酯的连续培养系统中生长的酵母中检测到，在 1 到 3 周的暴露时间内，诱变剂的数量与瓜氨酸抗性突变体菌落的数量之间存在明显的关系。对暴露于甲基甲磺酸酯的连续培养系统中生长的酵母进行全基因组测序表明，通过鉴定每个菌株中独特变体的数量，可以实现突变的定量。然而，这种方法的灵敏度低于平板测定法，并且无法区分不同浓度的诱变剂。总之，我们提出，在连续培养系统中生长的酵母可以提供一种改进的、更敏感的致突变性测试方法。
