Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.
Microbiology (Reading). 2022 Jun;168(6). doi: 10.1099/mic.0.001201.
, the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of as a counter-selectable marker that can be used for markerless mutagenesis in . encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in . We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and , that efficiently rendered susceptible to growth on 2-DOG. We then adapted the -based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for has a high success rate, and should be a valuable gene editing tool in the future.
淋病奈瑟菌是一种主要的公共卫生关注病原体，但该物种的基因操作方法还不够复杂。例如，几乎没有产生无标记突变的方法，而无标记突变可以在不引入额外 DNA 序列的情况下产生精确的点突变和缺失。无标记突变是研究发病机制、抗微生物药物耐药性（AMR）传播以及疫苗开发的核心。在这里，我们描述了使用 作为一种可选择的标记，可用于淋病奈瑟菌的无标记诱变。 编码半乳糖激酶，这是一种可以代谢能将其作为唯一碳源的细菌中的半乳糖的酶。GalK 还可以将半乳糖类似物 2-脱氧半乳糖（2-DOG）磷酸化为有毒的、不可代谢的中间产物 2-脱氧半乳糖-1-磷酸。我们利用 GalK 的这一特性，开发了一种用于淋病奈瑟菌无标记诱变的方法。我们成功地删除了对淋球菌疫苗开发和 AMR 传播研究都很重要的染色体和质粒编码基因。我们设计了一个基于抗生素抗性标记和 的正负选择盒，有效地使 对 2-DOG 的生长敏感。然后，我们适应了基于的反向选择和 2-DOG 的无标记诱变，并用生化和表型分析来确认靶基因的缺失。我们表明，我们用于 的无标记诱变方法成功率很高，将来应该成为一种有价值的基因编辑工具。
