Valdivia R H, Falkow S
Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305, USA.
Mol Microbiol. 1996 Oct;22(2):367-78. doi: 10.1046/j.1365-2958.1996.00120.x.
The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival. To study the behaviour of acid-inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted. Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus. We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu. These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enzymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock induction. This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid-inducible genes.
鼠伤寒沙门氏菌在小鼠巨噬细胞内存活和复制的能力取决于吞噬体的低pH值。对酸性液泡的这种需求表明，低pH值是细胞内存活所需基因转录的重要环境刺激因素。为了研究酸诱导基因在吞噬体环境中的行为，我们应用了一种称为差异荧光诱导（DFI）的新型富集策略，以筛选鼠伤寒沙门氏菌文库中在pH 4.5时上调的启动子。DFI利用荧光增强型绿色荧光蛋白（GFP）和荧光激活细胞分选仪（FACS）进行基因筛选。在存在诱导刺激（如低pH值）的情况下，使用FACS对携带与突变型GFP蛋白（GFPmut）融合的随机启动子的高荧光细菌克隆进行分选。然后在中性pH下扩增该群体，并分选荧光最少的群体。在有或没有诱导条件下对荧光和非荧光细菌进行连续分选，可快速富集受诱导刺激调控的启动子融合体。我们已经鉴定出八个酸诱导启动子，并对它们在pH 4.5和吞噬体环境下的表达进行了定量。这些酸诱导启动子与编码细胞表面维持酶、应激蛋白和通用外排泵的基因的启动子区域具有广泛的同源性。在巨噬细胞中，只有这些启动子的一个子集被诱导，其动力学和表达水平不一定与体外pH冲击诱导相关。这表明，虽然低pH值是细胞内基因表达的相关诱导剂，但巨噬细胞中的其他刺激因素可以调节酸诱导基因的表达。
