Abstract
Cyclic dinucleotides, including cyclic di-AMP (c-di-AMP), are known to be ubiquitous second messengers involved in bacterial signal transduction. However, no transcriptional regulator has been characterized as a c-di-AMP receptor/effector to date. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in Mycobacterium smegmatis, as a c-di-AMP receptor in bacteria. Ms5346 could specifically bind c-di-AMP with K(d) of 2.3 ± 0.5 μM. Using EMSA and DNase I footprinting assays, c-di-AMP was found to stimulate the DNA binding activity of Ms5346 and to enhance its ability to protect its target DNA sequence. A conserved 14-bp palindromic motif was identified as the DNA-binding site for Ms5346. Further, Ms5346 was found to negatively regulate expression of three target genes including Ms5347 (encoding a major facilitator family transporter), Ms5348 (encoding a medium chain fatty acyl-CoA ligase), and Ms5696 (encoding a cold shock protein, CspA). Ms5346 is the first cyclic di-AMP receptor regulator to be identified in bacteria, and we have designated it as DarR. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria.
Highlights
No transcriptional factor has been characterized as a c-di-AMP-responsive regulator in bacteria
We identified a TetR-like transcriptional factor, DarR, to be a bacterial c-di-AMP receptor regulator in M. smegmatis. c-di-AMP was found to stimulate the DNA binding activity of the DarR protein, which recognizes a 14-bp conserved sequence motif
It has become clear that cyclic dinucleotides can act as second messengers and play important roles in multiple bacterial physiological processes and even the interaction of bacterial pathogens with host cells [1,2,3,4,5,6]
Summary
No transcriptional factor has been characterized as a c-di-AMP-responsive regulator in bacteria. Conclusion: DarR is the first cyclic di-AMP receptor regulator to be identified in bacteria. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in Mycobacterium smegmatis, as a c-di-AMP receptor in bacteria. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria. C-di-AMP was found to be secreted by the pathogen Listeria monocytogenes and could stimulate an IFN--mediated host immune response [5] These findings suggested that c-di-AMP could act as either an intracellular or extracellular signal to participate in regulating bacterial physiology and pathogenesis. In this study, using a c-di-AMP/transcription factor binding screen, we have identified the first c-di-AMP receptor regulator, DarR (encoded by Ms5346), in M. smegmatis. Our findings provided an opportunity to understand the physiological function and regulatory mechanism of c-di-AMP
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