Towards understanding cyclic di-AMP metabolism

Bacteria rely on small signaling molecules to rapidly adapt to changes in their environment. One such molecule, cyclic di-AMP, has emerged over the past decade as an essential regulator of bacterial physiology. This thesis investigates how cyclic di-AMP is synthesized, broken down, and used by bacteria to maintain their viability in a dynamic external environment. Chapter 1 places cyclic di-AMP in a broader physiological context, showing that its primary role is to control bacterial cell volume. By regulating the movement of ions and small molecules across the cell membrane, cyclic di-AMP allows cells to maintain internal pressure and adapt to osmotic stress. The chapter then explores how other essential cell processes are connected to cyclic di-AMP signaling, for example, cell wall synthesis, DNA replication and the stringent response, and how they are connected to cell volume control. Chapter 2 focuses on the enzyme responsible for cyclic di-AMP synthesis, CdaA. Using a combination of biochemical and structural methods, chapter 2 demonstrates that CdaA may only synthesize cyclic di-AMP efficiently when membrane-embedded, where it forms an active complex. Chapter 3 examines how cyclic di-AMP is broken down. Chapter 3 focuses on NrnA and GdpP, enzymes that are required for breakdown of cyclic di-AMP to pApA (GdpP) and finally from pApA to AMP (NrnA). NrnA is an enzyme essential which maintains bacterial viability, but which does not directly degrade cyclic di-AMP. Instead, NrnA clears the potentially toxic accumulation of dinucleotide breakdown products, like pApA. Together, this thesis presents a clear and concise investigation into cyclic di-AMP signalling, followed by investigation of key cyclic di-AMP metabolic enzymes, and finishes by exploring the implications of cyclic di-AMP research in microbial physiology and future development of cyclic di-AMP-targeted antibacterial therapies.