Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571.

Yang Si,Bei Rao,Juanjuan Zhu,Dongsen Guo,Gaofei Jiang,Zengtao Zhong,Shuoxue Deng,Jun Zhu,Xiuming Lu,Yajun Cao,Daogeng Yu

doi:10.3390/genes11030335

Abstract

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

Highlights

Reactive oxygen species (ROS), such as hydroperoxides, organic peroxides, and free radicals, are continuously produced as by-products of various metabolic pathways
We have comprehensively studied several antioxidant enzymes in A. caulinodans, such as catalase (KatG) [17], alkyl hydroperoxide reductase (AhpC) [18], and bacterioferritin comigratory protein (BCP) [19], which are all involved in detoxifying exogenous H2 O2, and affect the nodulation behaviour and nitrogen fixation in S. rostrata [17,18,19]
Bioinformatics analyses of the A. caulinodans ORS571 genome were performed in order to study the organic peroxide resistance system in A. caulinodans

Summary

Introduction

Reactive oxygen species (ROS), such as hydroperoxides, organic peroxides, and free radicals, are continuously produced as by-products of various metabolic pathways. ROS are highly reactive and toxic [1,2], which causes damage to proteins, lipids, and DNA, eventually leading to cell death [3,4]. ROS can affect the interaction between microorganism and host [5], in microbes that are hypersensitive to ROS [6]. A type of aerobic Gram-negative bacteria, are able. Genes 2020, 11, 335 to reduce atmospheric nitrogen through a symbiotic interaction with a host legume [7]. During the early phases of legume-rhizobia symbiosis, plants generate ROS to defend against the invading species [9,10]

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Results

Conclusion