The canonical two-component systems (TCS) consist of a histidine kinase and a response regulator that work together to control various pathways in bacteria. Rhizobia are rod-shaped, Gram-negative alpha-proteobacteria capable of establishing a nitrogen-fixing symbiosis with compatible legume hosts. These bacteria can live freely in the soil or as intracellular symbionts within root nodules. Here, we characterized an orphan OmpR-type response regulator in Rhizobium etli CE3, which we renamed CenR due to its similarity to CenR proteins known as essential regulators of cell envelope-related functions in alpha-proteobacteria. We identified the cognate histidine kinase encoded by cenK, located in a separate genomic region from cenR. CenR and CenK form a TCS that has not been previously reported in Rhizobium. Our results indicate that the overexpression of cenR as well as the absence of cenK, negatively impacts R. etli growth and cell morphology, while bacteria overexpressing cenR also exhibit uncoordinated cell division. Furthermore, we demonstrated that the CenKR TCS directly or indirectly regulates the expression of essential genes involved in pathways that control cell growth and morphology. Electrophoretic mobility shift assays confirmed that CenR binds directly to the promoter regions of two uncharacterized genes in R. etli. Furthermore, analysis of the R. etli - common bean (Phaseolus vulgaris) symbiosis revealed increased infection threads, reduced leghemoglobin content, and lower nitrogen fixation efficiency in nodules infected by the cenR-overexpressing strain. In conclusion, our findings revealed that the CenKR TCS coordinates important cell cycle events in Rhizobium that are vital for both free-living and symbiotic conditions.

Keywords: CenR; Response regulator; Rhizobium etli symbiosis; Two-component system.

Copyright © 2025 The Authors. Published by Elsevier GmbH.. All rights reserved.

经典的双组分系统（TCS）由一个组氨酸激酶和一个响应调节器组成，二者共同控制细菌中的各种途径。根瘤菌是一种杆状、革兰氏阴性α-变形杆菌，在与相容的豆科植物宿主建立固氮共生关系方面具有能力。这些细菌可以在土壤中自由生活，也可以作为细胞内共生体存在于根瘤中。在这里，我们描述了在根瘤菌etli CE3中的一种孤儿OmpR型响应调节器，并将其重新命名为CenR，因为它与α-变形杆菌中的已知重要细胞壁相关功能调控因子CenR蛋白相似。我们确定了一个由cenK编码的同源组氨酸激酶，位于一个独立于cenR的基因组区域中。CenR和CenK形成了一种在根瘤菌中尚未报道过的TCS。我们的结果显示，cenR过表达以及缺乏cenK均对R. etli生长和细胞形态产生负面影响，而过表达cenR的细菌也表现出不协调的细胞分裂。此外，我们还证明了CenKR TCS直接或间接调节控制细胞生长和形态的关键基因的表达。电泳迁移率变动试验证实CenR可以直接结合到两个未表征的基因在R. etli中的启动子区域。此外，对根瘤菌-菜豆（Phaseolus vulgaris）共生关系的研究表明，在cenR过表达株感染形成的根瘤中，侵染线增加、豆血红蛋白含量降低以及固氮效率下降。总之，我们的研究发现揭示了CenKR TCS在协调Rhizobium中的重要细胞周期事件方面具有关键作用，这对自由生活和共生条件都至关重要。

关键词： CenR；响应调节器；根瘤菌etli共生关系；双组分系统。

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