Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR
Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR
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DOI：
10.1128/aem.01669-12
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发表时间：
2012-10-01
影响因子：
4.4
通讯作者：
Yoshida, Ken-ichi
中科院分区：
文献类型：
--
作者：
Suzuki, Hirokazu;Murakami, Ayano;Yoshida, Ken-ichi
反选择系统通过对将标记基因引入微生物和从微生物中消除标记进行正选择来促进微生物中的无标记遗传修饰。在此，我们报告了嗜土芽孢杆菌 HTA426 的反选择系统，该系统是通过同时破坏pyrF和pyrR基因而建立的。 PyrF 基因是嘧啶生物合成和 5-氟乳清酸 (5-FOA) 代谢为有毒代谢物所必需的，该基因因同源重组而被破坏。所得 MK54 菌株 (DeltapyrF) 是尿嘧啶营养缺陷型，并且对 5-FOA 具有抗性。与pyrF互补的MK54对尿嘧啶是原养型的，但在尿嘧啶存在的情况下对5-FOA不敏感。为了赋予 5-FOA 敏感性，编码衰减子的pyrR 基因被破坏，该衰减子通过感知尿嘧啶衍生物来抑制嘧啶生物合成。所得 MK72 菌株（DeltapyrF DeltapyrR）是尿嘧啶营养缺陷型，并且对 5-FOA 具有抗性。与pyrF互补的MK72对尿嘧啶是原养型的，并且即使在尿嘧啶存在的情况下也对5-FOA敏感。结果表明，pyrF 可以作为 MK72 中的反选择标记，这一点通过异源 β-半乳糖苷酶和 α-淀粉酶基因的有效无标记整合得到证明。整合的基因在嗜热G. kaustophilus 中功能性表达，并赋予嗜热菌新功能。该报告描述了在芽孢杆菌相关细菌中首次建立基于pyrF的反选择系统，以及首次在G. kaustophilus中证明同源重组和异源基因表达。我们的结果还提出了建立反选择系统的新策略。
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 (Delta pyrF) 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 (Delta pyrF Delta pyrR) 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 beta-galactosidase and a-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.