| Cyclic diadenosine monophosphate(c-di-AMP)is a recently discovered nucleotide second messenger.Various kinds of physiological functions have been found to be regulated by c-di-AMP,such as the balance of osmotic pressure,synthesis of fatty acid and transport of ions.The diadenylate cyclases(DAC),which contain a DAC domain and phosphodiesterases(PDE),which contain either a DHH-DHHA1 or HD domain,are c-di-AMP synthesis and degradation enzyme,respectively.c-di-AMP appears to exert its regulatory effects through changing the conformations of effector proteins or riboswitches.Thus c-di-AMP is able to regulate gene expression via both transcriptional and post-transcriptional control.Riboswitches are ligand-binding elements located in the 5'-untranslated regions or 3'-untranslated regions of mRNAs,which regulate expression of adjoining open reading frame(ORF).Through searching the Rfam database,four putative c-di-AMP riboswitches denoted as Btl,Bt2,Bt3 and Bt4 were identified in Bacillus thuringiensis BMB171.The downstream genes of Btl,Bt2,Bt3 and Bt4 encode hypothetical proteins,Kdp family proteins,amino acid permease and hypothetical protein,respectively.Sequence alignment revealed that the four riboswitches are widely distributed and conserved in the Bacillus cereus group.Next,the secondary structures of the four riboswitches were analyzed.Downstream the aptamer domain of Bt2 or Bt3,a Rho-independent terminator was identified,leading us to suspect that these two riboswitches may regulate the expression of downstream genes by forming a terminator or anti-terminator structure.However,terminator structures were not identified in Btl and Bt4.Therefore,some other unknown mechanisms may exist in controlling their downstream genes expression.To get a further insight into their regulation mechanisms,we have constructed expression vectors pHT1K-P,-lacZ(Px is P1?P2?P3 or P4,represents the promoter region containing Btl,Bt2,Bt3 or Bt4 respectively),and transformed them into BMB171 and?dis?cdaS strains.The results showed that the ?-galactosidase activities were increased in the lower c-di-AMP concentration strain ?disA?cdaS.Comparision of the RT-qPCR results in BMB171 and ?disA?cdaS strains also indicated that the transcription levels of downstream genes were increased in the ?disA?cdaS strain.Next,the vector pRP-Btx-lacZ(x is 1,2,3 or 4)and pET28b-disA were co-transformed into Escherichia coli BL21(DE3)strain.But the ?-galactosidase activity was decreased when c-di-AMP concentrations were elevated.Finally,the dual-luciferase reporter assay system further showed that the four riboswitches belong to the "off" switches.These results demonstrated that the four putative Btl,Bt2,Bt3 and Bt4 riboswitches can respond to c-di-AMP,and binding of this ligand inhibits the expression of downstream genes.Bt2 was selected for further study.The kdp operon is situated downstream of Bt2,and contains six structural genes of sipW,kdpF,kdpA,kdpB,kdpC and kdpD.RT-qPCR results revealed that the six genes were co-transcribed.In addition expression of kdp operon was found to be 10 times higher when the growth medium was supplemented with lower K+concentration(1 mM)than in higher K+concentration(30 mM),indicating that the expression of kdp operon could be induced by low K+ concentration.Further,the strains containing vectors pHT1K-P0-lacZ and pHT1K-P2-lacZ,respectively,were cultivated in high K+ and low K+ mediums.?-galactosidase activity revealed that the promoter of BMB171 kdp operon was not affected by different K+ concentration,and Bt2 could sense the K+ concentration to regulate the expression of kdp operon probably through forming a terminator or an anti-terminator structure.Under the low K+ condition,an anti-terminator structure was formed,and expressions of downstream genes were enhanced.On the other hand,under the high K+ condition,a Rho-independent terminator was formed,and downstream genes expressions were inihbited.Finally,a Bt2 in-frame deletion mutant strain ?Bt2 and a kdpD in-frame deletion mutant strain ?kdpD were constructed.The results of RT-qPCR showed that unlike the ?kdpD kdp operon,the ?Bt2 kdp operon could no longer respond to different K+ concentrations.Therefore,Bt2 could sense the different K+ concentration and regulate the expression of BMB171 kdp operon.Bt2 could thus respond to both K+ and c-di-AMP.Taken together,our research confirmed that the four putative c-di-AMP riboswitches were repressed by c-di-AMP in BMB171,and Bt2 could not only be regulated by c-di-AMP,but also by K+,which enrich the regulation mechanism of kdp operon. |
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