Identification Of The Protein That Binds To The Global Post-transcriptional Regulator RsmA In Xanthomonas Campestris PV.Campestris

RsmA(repressor of secondary metabolism A),in some bacteria called CsrA(Carbon storage regulator A),is widely distributed in various bacteria and acts as a global post-transcriptional regulator,playing a very important regulatory role in different physiological processes including central carbon metabolism,gluconeogenesis,glycogen synthesis,biofilm formation,flagella production,quorum sensing and pathogenesis.Our previous study found that,in Xanthomonas campestris pv.campestris(Xcc),RsmA acts as a global post-transcriptional regulator playing an important role in virulence,hypersensitive response,extracellular polysaccharide production,extracellular enzyme activity,cell motility,and biofilm formation.But the mechanism by which RsmA controlling these processes are mostly unclear.RsmA is a typical RNA-binding protein,and it has long been recognized that binding to its target mRNA is the only way for RsmA to exert its regulatory function.However,recent study found that RsmA can function by binding to target proteins.It is possible that RsmA also functioning via protein-protein interactions in Xcc.Based on this hypothesis,the aim of this study is to identify the protein that binds to RsmA in Xcc at genome-wide level by using the cross-linking and immune precipitation technique.To identify all RsmA-binding proteins(including transient binding and weak binding proteins),the RsmA-3F(a 3×Flag antigen Tag was fused at the C-terminus of the RsmA protein of Xcc)expressing strain,2506-3F,and the control strain Xcc8004(expressing the RsmA protein without any antigen Tag)were grown in the rich medium NYGB,thenXcc cells were treated with UV(cross-linking)to make RsmA covalently bound to its target proteins,and total proteins were extracted and the'RsmA-target protein' complex was isolated by co-immune precipitation(Co-IP)technique using 3×Flag magnetic beads.Finally,the protein composition of the precipitated'RsmA-protein complex'was determined by mass spectrometry and thus the proteins bound to RsmA were identified.As a result,76 proteins were identified in the 'RsmA-protein complex' sample isolated from the 2506-3F strain,while only 16 proteins from the control strain Xcc8004.After comparative analysis,48 proteins were identified as the RsmA-binding candidates.Interestingly,among the 48 candidate proteins,19 proteins are the enzymes in the central carbon metabolism pathway of Xcc,suggesting that RsmA regulates central carbon metabolism through protein-protein interactions.To further determine whether these 48 candidate proteins actually bind to RsmA in vivo,we used bacterial two-hybrid technique to validate the direct binding of some candidate proteins with RsmA.The results reveal that one protein,ProA(XC1880),strongly interacts,and 7 proteins[Conserved hypothetical protein(XC0932)?PpsA(XC1952)?RpfA(XC2329)?RpoA(XC3316)?RpsG(XC3344)?RpoB(XC3347)? RplA(XC3350)]weakly interact with RsmA in Xcc.To further confirm whether these eight proteins are indeed directly bound to RsmA,we further used mutual immune co-precipitation detect the in vivo interaction between RsmA and these proteins.The results demonstrate that ProA protein bind directly to RsmA in vivo.This study demonstrates that RsmA of Xcc can directly bind to ProA protein,indicating that RsmA in Xcc can also exert its regulatory function through protein-protein interaction.ProA is the third RsmA-binding protein after FliW and CesT.Our research broadens the understanding of the mechanism of RsmA action.