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Updated: Dec 25, 2025
A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues
Published on: February 19, 2019
Identification of a Streptococcus pyogenes SF370 gene involved in production of c-di-AMP
Taichi Kamegaya1, Kenji Kuroda, Yoshihiro Hayakawa
1Graduate School of Medicine, Nagoya University. kamegaya@med.nagoya-u.ac.jp
Abstract:
Here we show that bis(3'-5') cyclic diadenylic acid (c-di-AMP) and a diadenylate cyclase (DAC) domain protein involved in the biosynthesis of c-di-AMP were identified in Streptococcus pyogenes. The matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrum of the cell extract of S. pyogenes, which showed a fragment pattern very similar to that of the authentic sample of c-di-AMP, revealed that S. pyogenes produces c-di-AMP in the cell. Subsequently, we confirmed by an in vitro experiment that the production of c-di-AMP in the cell is due to the action of Spy1036 gene encoding a DAC domain protein named spyDAC, which is a new protein different from a well-known diadenylate cyclase. Moreover, the experiment gave a product with a molecular weight of 657.021, which is consistent with the molecular weight of c-di-AMP. Furthermore, the mass spectral fragment pattern of the product obtained by the in vitro biosynthesis is quite similar to that of the product produced by the above in vivo experiment. This in vitro production of c-di-AMP indicated that spyDAC in S. pyogenes actually catalyzes the in vivo biosynthesis of c-di-AMP from ATP.
Insights
Streptococcus pyogenes produces bis(3'-5') cyclic diadenylic acid (c-di-AMP). A novel diadenylate cyclase (DAC) protein, spyDAC, encoded by the Spy1036 gene, was identified as responsible for this c-di-AMP biosynthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bis(3"-5") cyclic diadenylic acid (c-di-AMP) is a crucial second messenger in bacteria.
- The biosynthesis pathways of c-di-AMP are not fully elucidated in all bacterial species.
Purpose of the Study:
- To identify and characterize the enzymes responsible for c-di-AMP biosynthesis in Streptococcus pyogenes.
- To investigate the role of a novel diadenylate cyclase (DAC) domain protein in S. pyogenes.
Main Methods:
- Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to detect c-di-AMP in S. pyogenes cell extracts.
- In vitro enzymatic assays were performed using the identified DAC domain protein (spyDAC) and ATP as substrate.
- Mass spectrometry was used to analyze the in vitro synthesized product.
Main Results:
- S. pyogenes was confirmed to produce c-di-AMP intracellularly.
- A novel DAC domain protein, spyDAC, encoded by the Spy1036 gene, was identified in S. pyogenes.
- In vitro experiments demonstrated that spyDAC catalyzes the biosynthesis of c-di-AMP from ATP, yielding a product with the correct molecular weight and mass spectral fragmentation pattern.
Conclusions:
- The Spy1036 gene product, spyDAC, is responsible for the in vivo biosynthesis of c-di-AMP in Streptococcus pyogenes.
- This finding reveals a new player in bacterial second messenger synthesis and provides a target for potential antimicrobial strategies.
