Martín-Mora David, Ortega Alvaro, Reyes-Darias José A, García Vanina, López-Farfán Diana, Matilla Miguel A, Krell Tino
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
Front Microbiol. 2016 Nov 29;7:1937. doi: 10.3389/fmicb.2016.01937. eCollection 2016.
is an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for α-ketoglutarate (αKG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) showed that it recognizes exclusively α-ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity ( = 301 μM, = 81 μM). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 μM and 5.9 μM were derived for ligand-free and αKG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated that showed chemotaxis to a broad range of αKG concentrations with maximal responses at 500 μM. Deletion of the gene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of αKG does not modulate expression. Since αKG is present in plant root exudates it was investigated whether the deletion of altered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for αKG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis toward tricarboxylic acid cycle intermediates.
是一种能感染人类、动物和植物的普遍存在的病原体。趋化作用被发现与这种病原体及其他病原体的毒力有关。尽管已被确立为趋化作用研究的模型，但26种化学感受器中的大多数在功能上仍未得到注释。我们在此报告将PA5072（命名为McpK）鉴定为α-酮戊二酸（αKG）的化学感受器。对重组McpK配体结合结构域（LBD）进行的高通量热迁移分析和等温滴定量热法研究（ITC）表明，它只识别α-酮戊二酸。ITC分析表明，配体以正协同性结合（Kd = 301 μM，Ka = 81 μM）。预测McpK具有螺旋双模块（HBM）类型的LBD，并且这项研究及其他研究表明，这种结构域类型可能与有机酸的识别有关。分析型超速离心（AUC）研究表明，McpK-LBD以单体-二聚体平衡形式存在。α-KG结合使二聚体稳定，对于无配体和αKG结合形式的McpK-LBD，二聚体自解离常数分别为55 μM和5.9 μM。对于其他含有HBM结构域的受体，已观察到配体诱导的LBD二聚体稳定，这可能对应于该蛋白家族的一种普遍机制。定量毛细管趋化分析表明，在500 μM时显示出对广泛浓度的αKG的趋化作用且有最大反应。基因缺失使整个浓度范围内的趋化作用降低至接近背景水平，互补后恢复了野生型样的趋化作用。实时PCR研究表明，αKG的存在不会调节表达。由于αKG存在于植物根系分泌物中，因此研究了基因缺失是否改变了玉米根系定殖。然而，未观察到相对于野生型菌株的显著变化。存在对αKG特异的化学感受器可能归因于其在中心代谢中的作用以及其作为信号分子的功能。这项工作扩展了已知化学感受器类型的范围，并强调了对三羧酸循环中间体趋化作用的重要生理作用。
