Organization of Advanced Science and Technology, Kobe University, Hyogo, Japan.
Appl Environ Microbiol. 2012 Oct;78(20):7376-83. doi: 10.1128/AEM.01669-12. Epub 2012 Aug 10.
Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.
反选择系统通过对微生物中标记基因的引入和标记的消除进行正向选择，促进微生物无标记基因遗传修饰。本研究通过同时敲除pyrF 和 pyrR 基因，建立了适用于 Geobacillus kaustophilus HTA426 的反选择系统。pyrF 基因是嘧啶生物合成所必需的，并且能够将 5-氟乳清酸（5-FOA）代谢为有毒代谢物，该基因通过同源重组被破坏。所得的 MK54 菌株（ΔpyrF）是尿嘧啶营养缺陷型，对 5-FOA 具有抗性。MK54 用 pyrF 互补后，对尿嘧啶表现出原养型，但在存在尿嘧啶的情况下对 5-FOA 不敏感。为了赋予 5-FOA 敏感性，破坏编码衰减子的 pyrR 基因，该衰减子通过感应尿嘧啶衍生物来抑制嘧啶生物合成。所得的 MK72 菌株（ΔpyrFΔpyrR）是尿嘧啶营养缺陷型，对 5-FOA 具有抗性。MK72 用 pyrF 互补后，对尿嘧啶和 5-FOA 均表现出原养型，即使在存在尿嘧啶的情况下也是如此。结果表明，pyrF 可作为 MK72 中的反选择标记，这通过异源β-半乳糖苷酶和α-淀粉酶基因的高效无标记整合得到了证明。整合的基因在 G. kaustophilus 中具有功能性表达，并赋予该嗜热菌新的功能。本研究首次在芽孢杆菌相关细菌中建立了基于 pyrF 的反选择系统，并首次证明了同源重组和异源基因在 G. kaustophilus 中的表达。我们的结果还为反选择系统的建立提供了一种新策略。
