Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function
Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function
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DOI：
10.1128/aem.01626-19
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发表时间：
2019-09
影响因子：
4.4
通讯作者：
Rita A. Luu;Rebecca A. Schomer;Ceanne N. Brunton;Richard Truong;A. Ta;W. A. Tan;J. Parales;Yu-Jing Wang;Yu-Wen Huo;Shuangjiang Liu;J. Ditty;V. Stewart;R. Parales
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作者：
Rita A. Luu;Rebecca A. Schomer;Ceanne N. Brunton;Richard Truong;A. Ta;W. A. Tan;J. Parales;Yu-Jing Wang;Yu-Wen Huo;Shuangjiang Liu;J. Ditty;V. Stewart;R. Parales
在细菌基因组编码的数千种化学感受器中，相对较少的已经被功能表征。更重要的是，虽然甲基接受趋化蛋白（细菌中存在的主要类型的化学感受器）很容易从生物信息学上识别出来，但目前还不可能预测什么化学物质会与特定的化学感受器结合。趋化性在生物降解以及宿主-病原体和宿主-共生体相互作用中发挥作用，但目前许多研究由于无法识别相关的趋化受体配体而受到限制。使用杂交受体和这个简单的大肠杆菌报告系统可以快速和敏感地筛选潜在的化学效应物。本研究选择的融合位点产生了高比例的功能性杂交种，这表明它可以广泛地用于测试系统发育不同样本的化学受体反应。考虑到土壤细菌对化学引诱剂的检测范围很广，杂交受体也可以作为灵敏的生物传感器。土壤细菌通过使用各种感觉系统感知和响应环境线索，以适应多样化和快速变化的环境条件。双组分系统是一种广泛存在于生命所有三个领域的信号转导系统，通常由传感器激酶和反应调节因子组成。许多双组分系统通过调节基因表达来响应环境刺激。细菌趋化系统是一种经过修改的双组分系统，具有额外的蛋白质组分和反应，而不是调节基因表达，涉及行为适应并导致朝向或远离化学刺激的净运动。土壤细菌通常在其基因组中编码有20到40个或更多的化学感受器。为了简化细菌化学感受器感知的化学效应（配体）的鉴定，我们将恶臭假单胞菌化学感受器的传感结构域与大肠杆菌NarX/NarQ硝酸盐传感器的信号结构域融合，构建了杂交传感蛋白。对潜在引诱剂的反应通过β-半乳糖苷酶测定来监测，使用大肠杆菌报告菌株，其中硝酸盐反应的narG启动子与lacZ融合。PcaY、McfR和NahY分别是芳香酸、三羧酸循环中间体和萘的趋化受体，它们构建的杂交受体对检测已知的引诱剂具有敏感性和特异性，并且在大肠杆菌中测量的β-半乳苷酶活性与天然恶臭杆菌菌株的趋化性实验结果具有良好的相关性。此外，杂交受体的筛选成功地鉴定了化学受体蛋白的新配体，并鉴定了6个检测丙酸盐的受体。在细菌基因组编码的数千种化学感受器中，相对较少的已经被功能表征。更重要的是，虽然甲基接受趋化蛋白（细菌中存在的主要类型的化学感受器）很容易从生物信息学上识别出来，但目前还不可能预测什么化学物质会与特定的化学感受器结合。趋化性在生物降解以及宿主-病原体和宿主-共生体相互作用中发挥作用，但目前许多研究由于无法识别相关的趋化受体配体而受到限制。使用杂交受体和这个简单的大肠杆菌报告系统可以快速和敏感地筛选潜在的化学效应物。本研究选择的融合位点产生了高比例的功能性杂交种，这表明它可以广泛地用于测试系统发育不同样本的化学受体反应。考虑到土壤细菌对化学引诱剂的检测范围很广，杂交受体也可以作为灵敏的生物传感器。
Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors. ABSTRACT Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by β-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ. Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the β-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate. IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.