Research On Regulation Of Protein RHE_RS01180 About Horizontal Gene Transfer Of Symbiotic Island In Rhizobium Etli

Azorhizobium caulinodans ORS571 can be symbiotic with Sesbania rostrata,nodulating on both root and stem,so that the nitrogen efficiency is so high.ICEAc,the symbiosis region,also called symbiotic island,maintains on the chromosome of ORS571,including nod loci involved in the synthesis and secretion of nod factors and some transfer genes and so on.ICEAc can be excised,cyclized and transferred to recipient cell through conjugation under specific environment stimuli.Previous studies indicate that ICEAc can be transferred to rhizobia belonging to other genus across distance,such as Mesorhizobium huakuii 7653R and 93,Sinorhizobium medicae USDA1037,which can nodulate on Sesbania rostrata obtaining ICEAc,enlarging the host range.However,not all the rhizobial strains can acquire ICEAc.In previous work,we took the Rhizobium etli CFN42 of Rhizobium that can’t acquire the ICEAc as the object,and screened the gene RHE_RS01180 that may play roles in the acquisition of ICEAc by inserting transposon into CFN42 genome.We found that protein RHE_RS01180 belongs to XRE family,which contains N terminal with HTH domain and C terminal with high variable region about regulation.We verified the function of RHE_RS01180 by deletion and complementation.The transfer frequency of ICEAc ranges from<10-12 to about 10-7 when gene RHE_RS01180 is deleted.In addition,we did research on RHE RS01175,the upstream gene of RHE_RS01180,but it does have no effect on transfer of ICEAc and no connection with RHE_RS01180.In this research,to obtain the genes that are regulated by protein RHE RS01180,the transeriptone profiles of wild type CFN42 and a △1180 mutant of stationary were assayed with RNA sequencing.After sequencing,we analyzed the datum.The gene levels were calculated using the RPKM method.False discovery rate≤0.001 and the absolute value of log2 ratio≥1 were used to identify differentially expressed genes(DEGs).Finally,in all the DEGs,we obtained 685 remarkable DEGs in the RHE RS01180 mutant including16 up-regulated and 669 down-regulated genes by comparison with wildtype strain CFN42.In recipient cell,we assumed that there are two mechanisms that block ICEAc entering,one is that protein RHE_RS01180 regulates related proteins of restriction and modification;another is that protein RHE_RS01180 regulates transferase.According to our hypotheses and the datum of RNA sequencing,we took gene RHE-RS29330(a nuclease)and gene RHE_RS28720(transferase/acyl-homoserine-lactone synthase,also called rail)as candidate genes.We found that gene RHE_RS29330 does not contribute to the acquisition of ICEAc in CFN42 and the horizontal gene transfer frequency is less than 10-12 by deletion;Also,we constructed the RHE_RS29330 translational fusion to understand the regulation of RHE RS01180,indicating that RHE_RS29330 is regulated by gene RHE RS01180.As for gene rail,the horizontal gene transfer frequency is aslo less than 10-12,we have not detected its effect on transfer frequency.In this reaesarch,by means of online BLAST,we found that the homologues of protein RHE_RS01180 are relatively universal in bacteria,and most of them appear in Proteobacteria.In Proteobacteria,especially in y-proteobacteria,the percentage is 84.24%;However,rarely in α-proteobacteria.Unimaginable,we have not found homologues of protein of RHE RS01180 in the rhizobia we have studied in laboratory including the self-accepting and non-accepting strains.Moreover,we found that gene RHE_RSO1180 and RHE_RS01175 are located on the defense island where the gene encoded BA14K protein is anchor gene.This defense island is about 20.4 kb,total 25 genes,including three adjacent transposases,two restriction proteins,one recombinase of phage integrase family,one DNA invertase and other functional genes.The structure is basically consistent with defense island,and it’s speculated that there are genes involved in horizontal transfer of symbiotic island on the defense island.Hence,it needs further research.Previous work indicates that strains which is self-accepting ICEAc,such as M.huakuii 93 is obtained the ability nodulating on the stem of Sesbania rostrata when acquiring the isalnd.However,CFN42 can not acquire the island ICEAc before deleting RHE_RS01180.To understand the difference of strains with different genetic background when they acquire the symbiotic island,we studied the nodulation ability of CFN42△1180::ICEAc datum showed that the strain doesn’t nodulate as expected.Then we detected the expression of nodA from CFN42/ORS571 with or without 20 μM Naringenin,data showed that two separate nod genes can express in CFN42△1180::ICEAC.Therefore,we speculated that the two nod clusters are independently expressed and interfere with each other.In addition,the genetic background of strain also has effect on the synthesis of nod culster.To achieve nodulation,we tried to increase mutation rate by deleted gene mutS to increase the mutation rate and select by plant to establish the experimental evolution system.In a word,we found that RHE_RS01180 participates in the horizontal transfer process of the symbiotic island through transcriptomics,bioinformatics and physiology.Besides,the genetic background of strains aslo has effect on the transfer and the gene expression of symbiotic island.