Functional Analysis Of Manganese Ion Transport Proteins In Deinococcus Radiodurans

As an important member of the extremist microbial family,Deinococcus radiodurans has strong resistance to environmental stress.More and more research results have shown that the adaptability of D.radiodurans to extreme environments stems from its super strong antioxidant capacity.The high ratio of manganese to iron accumulated in cells has always been considered to be one reason for its antioxidant mechanisms.D.radiodurans is a model organism for studying oxidative stress.Studying the related regulatory mechanism of manganese ions can not only explore the extreme resistance mechanism of D.radiodurans,but also expand the regulatory mechanism of bacteria on metal ions,and widely apply this characteristic.In this paper,we studied the functions and application of manganese ion transport-related proteins in D.radiodurans through studying the proteins that affect intracellular manganese ion level.First,through bioinformatics method we predicted and found the manganese ion transport-related proteins DR1187 and DRB0131 in D.radiodurans belonging to Ter C family.Then we compared them with the manganese ion efflux protein Mnt P in Escherichia coli and the protein Yce F belonging to Ter C family in Bacillus subtilis.Through predictive analysis,both DR1187 and DRB0131 have transmembrane regions.According to the hydrophilicity and hydrophobicity analysis,the transmembrane region was connected by hydrophilic peptide bonds.The difference in the number of transmembrane regions between these proteins may be because of the cells' different membrane structures.Finally,through multi-sequence alignment and homology modeling structure analysis,it was found that the proteins encoded by the two genes both had two very important aspartic acid sites in the Mnt P protein.Compared with the wild-type,the dr1187 gene deletion mutant showed decreased resistance to manganese ions and increased intracellular manganese ion content,indicating that DR1187 was involved in the absorption and transport of manganese ions in D.radiodurans.At the same time,the dr1187 gene deletion mutant showed enhanced resistance to ultraviolet light,hydrogen peroxide,and ionizing radiation,and the ROS content in the mutant was lower than that in the wild-type strain.The point mutation at D67 and D183 in DR1187 revealed that the point mutation resulted in intracellular enrichment of manganese ions and exhibited higher oxidative resistance than wild-type,indicating that D67 and D183 in DR1187 played important roles in their participation in manganese ion transport and cellular oxidative resistance.The structure of cell wall was also an important factor affecting the transport of metal ions.The intracellular manganese ion content was found to be increased by constructing the S-layer protein deletion mutant ?dr2577.The results showed that under the stress of lead and cadmium,the expression of manganese efflux gene was upregulated,and the mutant ?dr2577 released more manganese ions.Lead and cadmium are the main toxic heavy metals accumulated in rice,which can cause intracellular oxidative stress and produce large amounts of ROS.The rice seedlings were co-cultured with D.radiodurans,and the results of electron microscopy and bacterial fluorescence labeling showed that D.radiodurans colonized the outer layer of rice roots,and the mutant ?dr2577 could effectively improve the yellowing of rice seedlings,and the significant decreases in plant height,root length,dry biomass,and chlorophyll contents caused by cadmium or lead treatment.At the same time,the ?dr2577 mutant could adsorb more lead and cadmium,and improve the content of manganese ions and glutamic acid in rice and the activity of antioxidant enzymes by releasing intracellular manganese ions and glutamic acid,thereby reducing the accumulation of cadmium and lead in rice and relieving the oxidative stress caused by them.Taking together,we investigated the manganese ion transport-related genes in D.radiodurans and found that DR1187 was involved in the absorption and transport of manganese ions as well as its resistance to oxidative stress,and it was a potential manganese ion efflux protein.At the same time,it was found that the S-layer protein DR2577 affected the accumulation of intracellular manganese ions,and its mutant?dr2577 effectively alleviated the toxicity of cadmium and lead to rice under stress by efflux of antioxidant small molecules such as manganese ions.The results provide a basis for elucidating the manganese ion metabolism and the stress resistance mechanism of D.radiodurans,and expand the important role of extremophiles in alleviating heavy metal pollution of crops.