Abstract
Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.
Highlights
Bacteria have to adapt to changing conditions during their life cycle
In Bacillus subtilis, DisA delays sporulation on encountering DNA damage which is signaled by a decrease in the cellular c-di-AMP level. c-di-AMP has been found to regulate the biosynthesis of cell wall component in Staphylococcus aureus and Bacillus subtilis [8,14,15] and promotes virulence of Listeria monocytogenes [16]
We have shown that Rv3586 has the step process whereas Rv2837c can hydrolyze the two phosphoDAC activity and can synthesize c-di-AMP from ATP in a two- diester bonds of c-di-AMP sequentially, the first one resulting in linear 59-phosphate di adenosine nucleotide which is further hydrolyzed to 59-AMP
Summary
Bacteria have to adapt to changing conditions during their life cycle. They have developed different pathways to sense these changes and signal them for adaptation. Cyclic AMP (cAMP) and ppGpp are two of the common signaling molecules present in bacteria. CAMP regulates other cellular functions like flagellum biosynthesis, virulence, biofilm formation directly or indirectly whereas ppGpp is involved in quorum sensing, virulence etc [6,7]. Cyclic-di-GMP (c-di-GMP) and c-di-AMP, two dinucleotides in prokaryotes act as secondary molecules and have regulatory roles in bacterial cell cycle, adaptation and virulence [6,8,9,10]. Recent work has shown that c-di-GMP regulates processes like synthesis of adhesins and exopolysaccharide matrix components, virulence, cell cycle [12]. Same is true for cdi-GMP hydrolyzing proteins Proteins encoding for these two activities are present across the prokaryotes implying conservation of these pathways in bacteria. In Bacillus subtilis, DisA delays sporulation on encountering DNA damage which is signaled by a decrease in the cellular c-di-AMP level. c-di-AMP has been found to regulate the biosynthesis of cell wall component in Staphylococcus aureus and Bacillus subtilis [8,14,15] and promotes virulence of Listeria monocytogenes [16]
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