Utratna Marta, Cosgrave Eoin, Baustian Claas, Ceredig Rhodri, O'Byrne Conor
Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland.
Bioeng Bugs. 2012 Mar-Apr;3(2):93-103. doi: 10.4161/bbug.19476. Epub 2012 Mar 1.
A characteristic of the food-borne pathogen Listeria monocytogenes is its tolerance to the harsh conditions found both in minimally processed foods and the human gastrointestinal tract. This trait is partly under the control of the alternative sigma factor sigma B (σ(B)). To study the mechanisms that trigger the activation of σ(B) , and hence the development of stress tolerance, we have developed a fluorescent reporter fusion that allows the real-time activity of σ(B) to be monitored. The reporter, designated Plmo2230::egfp, fuses the strong σ(B)-dependent promoter from the lmo2230 gene (which encodes a putative arsenate reductase) to a gene encoding enhanced green fluorescence protein (EGFP). The reporter was integrated into the genomes of the wild-type strain L. monocytogenes EGD-e as well as two mutant derivatives lacking either sigB or rsbV. The resulting strains were used to study σ(B) activation in response to growth phase and hyperosmotic stress. The wild-type was strongly fluorescent in stationary phase or in cultures with added NaCl and this fluorescence was abolished in both the sigB and rsbV backgrounds, consistent with the σ(B)-dependency of the lmo2230 promoter. During sudden osmotic upshock (addition of 0.5 M NaCl during growth) a real-time increase in fluorescence was observed microscopically, reaching maximal activation after 30 min. Flow cytometry was used to study the activation of σ(B) at a population level by hyperosmotic stress during exponential growth. A strong and proportional increase in fluorescence was observed as the salt concentration increased from 0 to 0.9 M NaCl. Interestingly, there was considerable heterogeneity within the population and a significant proportion of cells failed to induce a high level of fluorescence, suggesting that σ(B) activation occurs stochastically in response to hyperosmotic stress. Thus the Plmo2230::egfp is a powerful tool that will allow the stress response to be better studied in this important human pathogen.
食源性病原体单核细胞增生李斯特菌的一个特点是它能耐受轻度加工食品和人类胃肠道中的恶劣环境。这一特性部分受替代σ因子σB（σ(B)）的控制。为了研究触发σ(B)激活以及由此产生应激耐受性的机制，我们构建了一种荧光报告融合体，可用于实时监测σ(B)的活性。该报告基因命名为Plmo2230::egfp，它将来自lmo2230基因（编码一种假定的砷酸盐还原酶）的强σ(B)依赖性启动子与编码增强型绿色荧光蛋白（EGFP）的基因融合。该报告基因被整合到野生型单核细胞增生李斯特菌EGD-e菌株以及两个分别缺失sigB或rsbV的突变衍生物的基因组中。所得菌株用于研究σ(B)在生长阶段和高渗应激响应中的激活情况。野生型菌株在稳定期或添加了NaCl的培养物中发出强烈荧光，而在sigB和rsbV背景下这种荧光消失，这与lmo2230启动子对σ(B)的依赖性一致。在突然的渗透压升高（生长过程中添加0.5 M NaCl）时，通过显微镜观察到荧光实时增加，30分钟后达到最大激活。流式细胞术用于研究指数生长期高渗应激在群体水平上对σ(B)的激活。随着盐浓度从0增加到0.9 M NaCl，观察到荧光强烈且成比例增加。有趣的是，群体中存在相当大的异质性，相当一部分细胞未能诱导出高水平的荧光，这表明σ(B)的激活是对高渗应激的随机反应。因此，Plmo2230::egfp是一种强大的工具，将有助于更好地研究这种重要人类病原体的应激反应。
