Screening and verification of substrate proteins of methionine sulfoxide reductase in rhizobia

Abstract:

Rhizobia would encounter oxidative stress of reactive oxygen species (ROS) in the process of infecting leguminous plants. Methionine-containing proteins are easy to be oxidized to methionine sulfoxide, leading to changes in protein structure and function. Methionine sulfoxide reductases (Msrs) can reduce methionine sulfoxide to methionine, restoring protein structure and function. We have identified four Msrs in the genome of Mesorhizobium huakuii 7653R that are involved in oxidative stress response, while the mechanism remains unclear. 【Objective】 To identify the substrates of four Msrs and elucidate the roles of the four Msrs in M. huakuii 7653R. 【Methods】 According to the methionine content, we determined the distribution of all the proteins in M. huakuii 7653R. Then, we used the online protein interaction prediction tools to predict the substrates of the four Msrs, and performed gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment for the predicted substrates. Finally, we verified the interaction between them by using the bacterial two-hybrid system. 【Results】 The methionine content followed the normal distribution, with most proteins in the middle and few proteins on both sides. Six antioxidant enzymes and six transcription factors were selected as the candidate substrates of the four Msrs. Finally, the bacterial two hybrid results showed that two antioxidant enzymes and five transcription factors can interact with the four Msrs to different degrees. 【Conclusion】 We provided the proof in illustrating the role of Msrs in the oxidative stress response to M. huakuii 7653R and provided a new idea for the research on the mechanism of rhizobia in response to ROS.

Key words:reactive oxygen species;methionine sulfoxide reductase;rhizobia;oxidative stress;bacterial two-hybrid system;interacting substrates