Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans, to eliminate the additional mutations present in the ATCC 24067A genetic background, demonstrated that the hypermutator phenotype of the pol3D270G mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.

中文翻译：

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在体外传代过程中，DNA聚合酶POL3的自发突变会导致隐球菌属物种出现超突变表型。

微生物的体外传代可以导致选择与其原始亲本菌株具有不同特性的微进化衍生物。一个很好表征的实例是在源自人类病原性真菌新隐球菌型菌株的系列菌株之间观察到的表型差异。在近亲隐球菌中报道了第二例，其中经过充分研究的分离株ATCC 24067（52D）在不同实验室进行了体外传代后改变了其表型特征。这些衍生物之一，ATCC 24067A，具有降低的毒力并且还表现出超突变体表型，其中突变率与野生型相比增加。在这项研究中，ATCC 24067谱系变化背后的分子基础是通过亲本和传代菌株基因组的下一代测序确定的。该分析导致鉴定出点突变，该点突变引起由POL3编码的DNA聚合酶δ亚基的核酸外切酶校对域内的D270G氨基酸取代。与POL3的互补性证实该突变是造成该菌株超突变表型的原因。新生球藻突变的再生，以消除ATCC 24067A遗传背景中存在的其他突变，证明pol3D270G突变体的超突变体表型在体外引起快速微进化，但不会导致毒力降低。