Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum

Abstract:

[Background] Cyclic diadenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced primarily within Gram-positive bacteria and is involved in cell growth, survival, stress resistance and many other aspects of bacterial physiology; however, there are few studies on c-di-AMP in lactic acid bacteria currently. [Objective] The gene of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum was cloned and efficiently heterologous expressed in Escherichia coli, and its biochemical activity in vitro was studied. [Methods] c-di-AMP in L. plantarum-YRA7 was detected by HLPC and ESI-MS using the cell extract. Then the c-di-AMP-synthesizing enzyme gene (lpDacA) was cloned from the genomic DNA of L. plantarum-YRA7 and the recombinant plasmid pET-28a-lpDacA was constructed. The recombination protein was successfully expressed in E. coli BL21(DE3), and purified by Ni-NTA affinity chromatography. Then enzymatic characteristics in vitro was studied. [Results] c-di-AMP was detected in L. plantarum-YRA7. The recombinant expression plasmid pET-28a-lpDacA was constructed and the purified recombinant protein was obtained. The biochemical activity study showed that the purified recombinant protein converted ATP into c-di-AMP in vitro. Its c-di-AMP-synthesizing activity was dependent on divalent metal ions and exhibited higher activity in the presence of Mg2+ at a basic pH. We also found that RHR motif is essential for the c-di-AMP-synthesizing activity and is the ATP binding site of lpDacA. [Conclusion] Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from L. plantarum will laid a solid foundation for future exploration on the physiological role of c-di-AMP in L. plantarum.

Key words:c-di-AMP, Lactobacillus plantarum, prokaryotic expression