Department of Restorative Dentistry, Oregon Health and Science University, Portland, Oregon, USA.
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA.
mSphere. 2023 Jun 22;8(3):e0068222. doi: 10.1128/msphere.00682-22. Epub 2023 Apr 24.
The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog -chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.
变形链球菌遗传系统提供了多种策略，可快速对靶向染色体突变进行工程改造。以前，我们报道了第一个在野生型背景下起作用的变形链球菌负选择系统。该系统利用对有毒氨基酸类似物 - 对氯苯丙氨酸（4-CP）的诱导敏感性作为负选择机制，并开发用于基于反选择的无标记诱变（CIMM）。虽然我们已经广泛地将该系统用于我们正在进行的遗传研究，但我们遇到了该系统的一些限制，主要是其宿主范围狭窄和需要在有毒底物上进行选择。在这里，我们报告了一种新的负选择系统的开发，该系统解决了这两个限制，同时仍然保留了以前基于 4-CP 的无标记诱变系统的实用性。我们将各种毒素编码基因置于木糖诱导型基因表达盒（Xyl-S）的控制下，发现 Fst-sm 和 ParE 毒素是诱导性负选择的合适候选物。我们将诱导型毒素与抗生素抗性基因结合，创建了几种不同的反选择盒。其中最广泛使用的包含一个与 Xyl-S 表达系统的点突变形式转录融合的野生型开放阅读框，我们随后将其命名为 IFDC4。IFDC4 表现出异常低的背景抗性，当在添加木糖的培养基上进行平板培养时，观察到细胞数量减少了 3-4 个对数级。IFDC4 还在多种变形链球菌菌株以及链球菌和血链球菌中具有相似的功能。我们使用 IFDC4 进行 CIMM，并成功地在所有三种物种中设计了各种不同类型的无标记突变。这里描述的反选择策略提供了一个模板方法，应该可以适应许多其他细菌中类似反选择系统的创建。许多具有重要医学意义的链球菌种，如变形链球菌，具有高度复杂的遗传系统，这主要是由于它们易于通过自然转化进行遗传操作。尽管如此，在链球菌中创建无标记突变的选择很少，特别是在野生型菌株中。无标记诱变是遗传研究的关键工具，因为它允许用户探索许多使用标记诱变难以解决的基本问题。在这里，我们描述了一种新的链球菌无标记诱变方法，该方法与当前使用有毒底物 4-CP 诱导敏感性的方法相比具有多种优势。这里采用的方法应该可以很容易地适应其他生物体中类似无标记诱变系统的创建。
