Functional Characterization of a Small Alarmone Hydrolase in Corynebacterium glutamicum.
Functional Characterization of a Small Alarmone Hydrolase in Corynebacterium glutamicum.
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DOI：
10.3389/fmicb.2018.00916
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发表时间：
2018
影响因子：
5.2
通讯作者：
Persicke M
中科院分区：
文献类型：
--
作者：
Ruwe M;Rückert C;Kalinowski J;Persicke M
(pp)pGpp 代谢是细菌生理学的重要组成部分，因为它涉及各种应激反应和细胞稳态机制，例如生长调节。然而，为了更好地理解(pp)pGpp相关调控，研究(pp)pGpp代谢的分子机制至关重要。近年来，对 RelA/SpoT 同源物 (RSH) 超家族的生物信息学分析除了众所周知的双功能 Rel 蛋白之外，还发现了小型单功能 RSH 衍生物。这些也称为小警报酮合成酶 (SAS) 或小警报酮水解酶 (SAH)。在这项研究中，基于相应氨基酸序列与(pp)pGpp水解结构域的高度相似性，来自谷氨酸棒杆菌的ORF cg1485被鉴定为推定的SAH编码基因。其基因产物（称为 RelHCg）的表征代表了对 SAH 亚科细菌代表的首次功能研究。预测的焦磷酸水解酶活性通过两种大肠杆菌菌株（其特征在于不同的警报酮基础水平）中的表达以及纯化蛋白的体外分析在体内得到证实。在对三种已知警报酮种类的水解活性进行基于测定的分析过程中，发现 RelHCg 和双功能 RSH 酶 RelCg 对浓度超过 0.75 mM 的警报酮表现出明显的底物抑制作用。 (pp)pGpp 水解酶的这一特性可能是实现 (pp)pGpp 基础水平和应激相关警报酮产生之间的 (pp)pGpp 代谢双稳态特征的重要机制。 relHCg 的缺失仅对野生型背景和(pp)pGpp 合成酶缺失的缺失突变体的生长行为造成很小的影响。根据这一观察，该蛋白质可能仅在特定环境条件下存在或活跃。通过生物信息学分析发现，棒状杆菌属的许多代表中相应基因的独立丢失也支持了这一假设。此外，对三个活性谷氨酸棒杆菌RSH基因的所有可能缺失组合的生长分析揭示了有趣的功能关系，未来必须对其进行更详细的研究。
The (pp)pGpp metabolism is an important component of bacterial physiology as it is involved in various stress responses and mechanisms of cell homeostasis, e.g., the regulation of growth. However, in order to better understand the (pp)pGpp associated regulation, it is crucial to study the molecular mechanisms of (pp)pGpp metabolism. In recent years, bioinformatic analyses of the RelA/SpoT homolog (RSH) superfamily have led to the discovery of small monofunctional RSH derivatives in addition to the well-known bifunctional Rel proteins. These are also referred to as small alarmone synthetases (SASs) or small alarmone hydrolases (SAHs). In this study, the ORF cg1485 from C. glutamicum was identified as a putative SAH encoding gene, based on a high similarity of the corresponding amino acid sequence with the (pp)pGpp hydrolysis domain. The characterization of its gene product, designated as RelHCg, represents the first functional investigation of a bacterial representative of the SAH subfamily. The predicted pyrophosphohydrolase activity was demonstrated in vivo by expression in two E. coli strains, characterized by different alarmone basal levels, as well as by in vitro analysis of the purified protein. During the assay-based analysis of hydrolysis activity in relation to the three known alarmone species, both RelHCg and the bifunctional RSH enzyme RelCg were found to exhibit a pronounced substrate inhibition for alarmone concentrations of more than 0.75 mM. This characteristic of (pp)pGpp hydrolases could be an important mechanism for realizing the bistable character of the (pp)pGpp metabolism between a (pp)pGpp basal level and stress-associated alarmone production. The deletion of relHCg caused only a minor effect on growth behavior in both wild-type background and deletion mutants with deletion of (pp)pGpp synthetases. Based on this observation, the protein is probably only present or active under specific environmental conditions. The independent loss of the corresponding gene in numerous representatives of the genus Corynebacterium, which was found by bioinformatic analyses, also supports this hypothesis. Furthermore, growth analysis of all possible deletion combinations of the three active C. glutamicum RSH genes revealed interesting functional relationships which will have to be investigated in more detail in the future.