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

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.

Sinorhizobium meliloti ohrR genes affect symbiotic performance with alfalfa (Medicago sativa)

“…Detoxification of organic peroxides is important for establishment of Rhizobium ‐legume symbiosis, which is confirmed by the data from an A . caulinodans ohr mutant (Si et al ., 2020). Sinorhizobium meliloti mutants of the cpo gene and its regulator gene ohrR ( ohrR1 ) did not exhibit symbiotic deficiency after interactions with alfalfa, possibly owing to gene redundancy (Barloy‐Hubler et al ., 2004).…”

“…A chloroperoxidase (Cpo) and an organic hydrogen resistance protein (Ohr) are associated with tolerance of freeliving S. meliloti cells to organic peroxides, but the single-gene mutants lacked observable symbiotic defects, possibly as a consequence of gene redundancy (Barloy-Hubler et al, 2004;Fontenelle et al, 2011). The ohr deletion mutant of Azorhizobium caulinodans induced fewer stem nodules with low nitrogenase activity on Sesbania rostrata (Si et al, 2020), suggesting that detoxification of organic peroxides is vital for rhizobial nodulation symbiosis.…”

OhrR is a central transcriptional regulator of virulence in Dickeya zeae

“…The roles of OhrR in sensing and responding to oxidative stress are widely conserved in many bacteria ( Agrobacterium tumefaciens , Azorhizobium caulinodans , Bacillus subtilis , D. dadantii , Mycobacterium smegmatis , Pseudomonas aeruginosa , S. avermitilis , X. campestris ) (Atichartpongkul et al, 2016 ; Chandrangsu et al, 2018 ; Chuchue et al, 2006 ; Gaballa et al, 2014 ; Garnica et al, 2017 ; Grenier et al, 2006 ; Liu et al, 2016 ; Panmanee et al, 2002 ; Reverchon et al, 2010 ; Si et al, 2020 ). At the same time, OhrR has been found to be a key regulator of virulence in some pathogenic bacteria (Atichartpongkul et al, 2010 ; Das et al, 2019 ; Pande et al, 2018 ; Previato‐Mello et al, 2017 ).…”

Redox Regulation in Diazotrophic Bacteria in Interaction with Plants

“…A. caulinodans possesses the catalase-peroxidase KatG and the alkylhydroperoxide reductase AhpCD to cope with H 2 O 2 increase, which are differentially used depending on the growth phase and H 2 O 2 concentration [69,70]. In addition, protection against organic hydroperoxide stress requires the Ohr protein [71]. The katG, ahpC, and ohr single mutants displayed a significant reduction in stem nodule number associated with a reduced nitrogen fixation ability.…”