Functional Analysis Of TerD,A C-di-AMP Candidate Receptor Protein In Bacillus Thuringiensis BMB171

Cyclic diadenosine monophosphate（c-di-AMP）is a new member of bacterial cyclic nucleotide messengers which is firstly discovered in 2008.Under the stimulus of upstream signal molecules,the concentration of c-di-AMP in bacteria is mainly controlled by DACs（diadenylate cyclase）and PDEs（phosphodiesterase）directly;aDAC containing the DisA＿N domain catalyzes the condensation of two molecules of ATP to one c-di-AMP,while aPDE containing the DHH-DHHA1 or the HD domain degrades c-di-AMP into pApA or AMP.As a second messenger molecule,c-di-AMP usually targets a specific receptor or effector to regulate the expression of downstream genes.It is reported that c-di-AMP is involved in the following physiological processes:1)DNA integrity sensing,DNA damage repair and the regulation of endospore formation in Bacillus subtilis,2)synthesis of peptidoglycan,3)potassium ion transport,4)metabolism of fatty acid,and 5)other physiological functions in bacteria.However,the receptors and targets of these c-di-AMP modulating signaling pathways are still unknown.In this study,to isolate c-di-AMP-binding proteins from Bacillus thuringiensis BMB171,biotinylated c-di-AMP was coupled to streptavidin agarose resin,then we successfully established an affinity chromatography system concerning c-di-AMP as ligand.Using this c-di-AMP affinity column,we identified TerD₂ and TerD₃ as the potential candidate receptors of c-di-AMP.TerD is annotated as a stress response protein in BMB171;the crystal structure of TerD shows that TerD occupies a putative cAMP binding domain（maybe a c-di-AMP binding site）.Since TerD₁,TerD₂ and TerD₃ have the highly similarity with each other,and they locate in the same operon,all of them are proposed to be c-di-AMP receptors.In the following experiments,1)we conducted ³²P labeled isotope experiments,and found that TerD₁,TerD₂,and TerD₃ could bind both cAMP and c-di-AMP,while the binding of TerD₁,TerD₂,and TerD₃ to c-di-AMP is iiiccompanied by the presense of Ca²⁺,and we supposed that the binding of TerD₁,TerD₂,and TerD₃ to cAMP is stronger that that of c-di-AMP;2)LC-TOF/MS experiments indicated that TerD₃ could bind with cAMP,but not c-di-AMP;3)ITC experiments demonstrated that TerD₂ could interact with c-di-AMP,but not cAMP.At the same time,we successfully constructed a series of terD related gene knockout mutant strains by homing endonucleases I-Sce I mediated markerless gene replacement in BMB171,includingΔterD₃,ΔterD_2-3,ΔterD_1-3,ΔterC,ΔterD_1-3ΔterC,ΔyceG,ΔtelA,ΔyceGΔtelA and Δfl.Then we explored the physiological function of terD related gene in BMB171 through the study of phenotypes of these terD related gene knockout mutants.Phenotypes mainly contains motility,biofilm formation,high sodium,potassium,temperature and ultraviolet sensitivity,the influence of spore and parasporal crystal formation and bacterial tellurite resistance,etc.The experimental results demonstrate that other than bacterial tellurite resistance and temperature,motility,the formation of biofilms and other phenotypes showed no significant changes,In the bacterial tellurite resistance experiments,all the results of minimum in hibition concentration,disk diffusion assays and the growth inhibition experiment showed that the terD related gene deleted strains exhibited enhanced sensitivity of K₂TeO₃,which is consistent with pertinent literatures.Meanwhile,the missing of terD related gene could enhance the sensitivity of strain to low temperature and the resistance to high temperature.Our results show that TerD might be the receptor of cAMP and/or c-di-AMP,which enrichs the metabolic regulation network of cAMP and c-di-AMP,and lays the foundation of further exploration of tellurite resistance mechanisms in bacteria.