Genetic screening for bacterial mutants in liquid growth media by fluorescence-activated cell sorting
Genetic screening for bacterial mutants in liquid growth media by fluorescence-activated cell sorting
复制标题
DOI：
10.1016/j.mimet.2010.11.003
复制
发表时间：
2011-01-01
影响因子：
2.2
通讯作者：
Withey, Jeffrey H.
中科院分区：
文献类型：
--
作者：
Abuaita, Basel H.;Withey, Jeffrey H.
许多细菌病原体已经在液体培养基中定义了体外毒力诱导条件，其导致在感染期间重要的毒力因子的产生。鉴定不再对毒力诱导条件作出反应的突变体将增加我们对细菌发病机理的理解。然而，传统的遗传筛选需要在固体培养基上生长。单个菌落中的细菌处于生长曲线的每个阶段，这使分析复杂化，并使筛选生长阶段特异性突变体成为问题。在这里，我们利用荧光激活的细胞分选结合随机转座子诱变分离细菌生长在液体培养基中，是有缺陷的毒力激活。该方法允许在真实的时间内分析整个细菌群体，并在诱导后的任何时间点选择具有所需基因表达谱的单个细菌突变体。我们已经使用这种方法来鉴定霍乱弧菌的致病性诱导缺陷突变体。(C)2010 Elsevier B.V.保留所有权利。
Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and makes screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. (C) 2010 Elsevier B.V. All rights reserved.