Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species
Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species
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DOI：
10.1016/j.anaerobe.2016.09.004
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发表时间：
2016-12-01
期刊：
影响因子：
2.3
通讯作者：
Kuwahara, Tomomi
中科院分区：
文献类型：
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作者：
Kino, Yasuhiro;Nakayama-Imaohji, Haruyuki;Kuwahara, Tomomi
由于某些细菌的抗生素抗性标记数量有限，因此无标记基因缺失对于多基因破坏是必要的。然而，即使在可转化的菌株中，在没有标记的情况下获得预期的突变也需要对大量菌落进行费力的筛选。先前的研究在各种细菌中成功地使用了反向选择系统，其中将条件致死基因并入载体中。我们研究了突变的pheS基因（pheS*）作为拟杆菌中基因缺失的反选择性标记的功效。该突变在拟杆菌苯丙氨酰tRNA合成酶的α亚基中产生氨基酸取代（A303 G），其在大肠杆菌中改变tRNA合成酶的特异性，导致由于对氯苯丙氨酸（p-Cl-Phe）掺入蛋白质中而导致的条件性致死突变。B。fragilis YCH 46和B.在液体限定基本培养基（DMM）中和在DMM琼脂平板上，在>5 mM p-Cl-Phe的存在下，用携带pheS* 的穿梭载体转化的多形细胞VPI-5482的生长明显受到抑制。构建靶向质粒以缺失B中荚膜多糖PS 2的遗传区域。fragilis或PSI在B中。太多了。在反选择后，对于B，以8.1 × 10（-3）的频率产生p-Cl-Phe抗性菌落。fragilis和B为1.7 × 10（-3）。太多了。在p-Cl-Phe抗性菌落中，4.2%和72%具有B的正确遗传缺失。fragilis和B.分别为：这些结果表明，突变pheS是一个有用的反选择性基因构建无标记的遗传缺失类杆菌。(C)2016爱思唯尔有限公司版权所有
Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E.coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-CI-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PSI in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 x 10(-3) for B. fragilis and 1.7 x 10(-3) for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides. (C) 2016 Elsevier Ltd. All rights reserved.