Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells
Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells
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DOI：
10.1128/aem.69.12.7480-7491.2003
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发表时间：
2003-12-01
影响因子：
4.4
通讯作者：
Hinton, JCD
中科院分区：
文献类型：
--
作者：
Hautefort, I;Proença, MJ;Hinton, JCD
我们开发了一种可靠且灵活的基于绿色荧光蛋白 (GFP) 的系统，用于测量单个细菌细胞中的基因表达。到目前为止，大多数系统都依赖于质粒携带的 gfp 基因融合，存在与质粒不稳定相关的问题。我们证明最近开发的 GFP 变体 GFP(+) 适用于评估细菌基因表达。构建了各种gfp(+)转录融合体，并将其作为单拷贝整合到鼠伤寒沙门氏菌的染色体中。 proU-lacZ 和 proU-gfp(+) 融合体表达水平的比较表明，GFP(+) 报告的单个沙门氏菌细胞中的 proU 活性与 β-半乳糖苷酶报告的整个群体的活性一样准确。单拷贝 gfp(+) 融合体非常适合监测沙门氏菌毒力基因的上调和下调。我们发现 SPI1 基因 prgH 的体外诱导仅发生在部分群体中，并且比例随生长阶段而变化。我们测定了海洋巨噬细胞释放的细菌中 SPI2 基因 ssaG 的表达水平。我们的结果首次证明单拷贝 GFP(+) 融合体可以在简单和复杂的环境中可靠地报告基因表达。这种方法有望在动物感染期间准确测量单个细菌的基因表达。
We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP(+), is suitable for assessing bacterial gene expression. Various gfp(+) transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp(+) fusions showed that GFP(+) reported proU activity in individual Salmonella cells as accurately as beta-galactosidase reported activity for entire populations. The single-copy gfp(+) fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1 gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from marine macrophages. Our results demonstrate for the first time that single-copy GFP(+) fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.