Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Doctoral Program in Biophysics, University of Wisconsin-Madison, Madison, Wisconsin, USA.
mSystems. 2024 Jun 18;9(6):e0005024. doi: 10.1128/msystems.00050-24. Epub 2024 May 15.
The ability to modify and control natural and engineered microbiomes is essential for biotechnology and biomedicine. Fungi are critical members of most microbiomes, yet technology for modifying the fungal members of a microbiome has lagged far behind that for bacteria. Interdomain conjugation (IDC) is a promising approach, as DNA transfer from bacterial cells to yeast enables modification. While such genetic transfers have been known to naturally occur in a wide range of eukaryotes and are thought to contribute to their evolution, IDC has been understudied as a technique to control fungal or fungal-bacterial consortia. One major obstacle to the widespread use of IDC is its limited efficiency. In this work, we manipulated metabolic and physical interactions between genetically tractable and to control the incidence of IDC. We test the landscape of population interactions between the bacterial donors and yeast recipients to find that bacterial commensalism leads to maximized IDC, both in culture and in mixed colonies. We demonstrate the capacity of cell-to-cell binding via mannoproteins to assist both IDC incidence and bacterial commensalism in culture and model how these tunable controls can predictably yield a range of IDC outcomes. Furthermore, we demonstrate that these controls can be utilized to irreversibly alter a recipient yeast population, by both "rescuing" a poor-growing recipient population and collapsing a stable population via a novel IDC-mediated CRISPR/Cas9 system.IMPORTANCEFungi are important but often unaddressed members of most natural and synthetic microbial communities. This work highlights opportunities for modifying yeast microbiome populations through bacterial conjugation. While conjugation has been recognized for its capacity to deliver engineerable DNA to a range of cells, its dependence on cell contact has limited its efficiency. Here, we find "knobs" to control DNA transfer, by engineering the metabolic dependence between bacterial donors and yeast recipients and by changing their ability to physically adhere to each other. Importantly, we functionally validate these "knobs" by irreversibly altering yeast populations. We use these controls to "rescue" a failing yeast population, demonstrate the capacity of conjugated CRISPR/Cas9 to depress or collapse populations, and show that conjugation can be easily interrupted by disrupting cell-to-cell binding. These results offer building blocks toward mycobiome editing, with significant implications for clinical treatments of fungal pathogens and other fungal system engineering.
修饰和控制自然和工程微生物组的能力对于生物技术和生物医学至关重要。真菌是大多数微生物组的关键成员，但修饰微生物组中真菌成员的技术远远落后于细菌。种间共轭（IDC）是一种很有前途的方法，因为将细菌细胞中的 DNA 转移到酵母中可以实现修饰。虽然这种基因转移在广泛的真核生物中自然发生，并且被认为有助于它们的进化，但作为控制真菌或真菌-细菌共生体的技术，IDC 的研究还很有限。IDC 广泛应用的一个主要障碍是其效率有限。在这项工作中，我们操纵了可遗传的酵母细胞的代谢和物理相互作用，以控制 IDC 的发生率。我们测试了细菌供体和酵母受体之间种群相互作用的范围，发现细菌共生导致 IDC 最大化，无论是在培养物中还是在混合菌落中。我们证明了通过甘露糖蛋白进行细胞间结合的能力可以在培养物和模型中辅助 IDC 发生率和细菌共生，并且可以预测这些可调控制如何可预测地产生一系列 IDC 结果。此外，我们证明这些控制可以用于通过细菌共轭不可逆地改变受体酵母群体，通过“挽救”生长不良的受体群体并通过新型 IDC 介导的 CRISPR/Cas9 系统使稳定群体崩溃。
真菌是大多数自然和合成微生物群落中重要但经常被忽视的成员。这项工作强调了通过细菌共轭修饰酵母微生物组种群的机会。虽然共轭已被认为具有将可工程化的 DNA 递送到一系列细胞的能力，但它对细胞接触的依赖限制了其效率。在这里，我们通过设计细菌供体和酵母受体之间的代谢依赖性以及改变它们相互物理附着的能力来找到控制 DNA 转移的“旋钮”。重要的是，我们通过不可逆地改变酵母种群来验证这些“旋钮”的功能。我们使用这些控制来“挽救”失败的酵母种群，证明共轭 CRISPR/Cas9 的能力可以降低或崩溃种群，并表明共轭可以通过破坏细胞间结合轻松中断。这些结果为真菌组编辑提供了构建模块，对真菌病原体的临床治疗和其他真菌系统工程具有重要意义。
