Bacterial cell volume regulation and the importance of cyclic di-AMP

Abstract

SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and metabolic adaptations, often in response to environmental fluctuations of physical parameters. In the last two decades, cyclic di-AMP has emerged as an important signaling nucleotide in many prokaryotic lineages, including Firmicutes, Actinobacteria, and Cyanobacteria. Its importance is highlighted by the fact that both the lack and overproduction of cyclic di-AMP affect viability of prokaryotes that utilize cyclic di-AMP, and that it generates a strong innate immune response in eukaryotes. In bacteria that produce the second messenger, most molecular targets of cyclic di-AMP are associated with cell volume control. Besides, other evidence links the second messenger to cell wall remodeling, DNA damage repair, sporulation, central metabolism, and the regulation of glycogen turnover. In this review, we take a biochemical, quantitative approach to address the main cellular processes that are directly regulated by cyclic di-AMP and show that these processes are very connected and require regulation of a similar set of proteins to which cyclic di-AMP binds. Altogether, we argue that cyclic di-AMP is a master regulator of cell volume and that other cellular processes can be connected with cyclic di-AMP through this core function. We further highlight important directions in which the cyclic di-AMP field has to develop to gain a full understanding of the cyclic di-AMP signaling network and why some processes are regulated, while others are not.

Keywords: cell volume regulation; cell wall metabolism; cyclic di-AMP; osmoregulation; potassium and compatible solute transport; second messenger; signaling nucleotide.

Fig 1

Cell volume regulation in response to osmotic fluctuations. (A) Schematic representation of the bacterial response to osmotic stress. (B) Overview of the bacterial osmolyte influx and efflux systems. Gray circles = cell wall; blue circles = cell membrane; red squares = cyclic di-AMP; open circles = osmolytes; gray arrows = turgor pressure; dashed arrows = movement of water; yellow star = phosphorylated site. Protein domains: green = RCK_C; light blue = CBS; Purple = USP.

Fig 2

The effects of osmotic downshift and cell wall damaging antibiotics on cyclic di-AMP mutant strains. (A) Schematic representation of the bacterial response to the presence of cell wall damaging β-lactam antibiotics. Gray circles = cell wall; dashed gray circles = damaged cell wall; blue circles = cell membrane; red squares = cyclic di-AMP; open circles = osmolytes; gray arrows = turgor pressure; dashed arrows = movement of water. The increase/decrease in turgor pressure is inferred from lysis phenotype experiments and the fact that osmolyte transporters are regulated by cyclic di-AMP. (B) Overview of the bacterial components that relate cyclic di-AMP to cell wall metabolism. This includes cyclic di-AMP synthesis, efflux and extracellular breakdown systems, and cell wall synthesis machinery. The term yda0 refers to the cyclic di-amp responsive riboswitch, which in some cases is located before cell wall remodeling enzymes.

Fig 3

The roles of cyclic di-AMP during cell division and sporulation. (A) Overview of the bacterial cyclic di-AMP synthesis and effector proteins that are associated with cell division and/or sporulation. CdaA and DisA = cyclic di-AMP synthase; RadA = branch migration transferase; RecA = multifunctional protein involved in homologous recombination. (B) Schematic representation of the effect of inhibited cyclic di-AMP synthesis on cell division, that is,. when DisA encounters damaged DNA. FtsZ = Cell division protein involved in septum formation; Gray circles = cell wall; blue circles = cell membrane; red squares = cyclic di-AMP; open circles = osmolytes; gray arrows = turgor pressure; brown arrows = DNA. See the legend of Fig. 2 for turgor pressure predictions.

Fig 4

Comparison of the tricarboxylic acid cycle in different Firmicutes. (A) Schematic representation of the complete TCA cycle. (B) Comparison of the TCA cycle of B. subtilis (complete), L. monocytogenes (lacks the enzyme α-ketoglutarate), and L. lactis (lacks the enzyme isocitrate dehydrogenase). Red crosses = gene not present in the genome.

Fig 5

Connections between cyclic di-AMP and (p)ppGpp. Gray circle = cell wall; blue circle = cell membrane; GdpP, PgpH, NrnA = cyclic di-AMP phosphodiesterase; Rel = (p)ppGpp synthase/hydrolase; DarB/CnpB = cyclic di-AMP-binding protein and regulator of Rel.

Fig 6

The effect of different nucleotides on the regulatory function of SbtB in cyanobacteria during light and dark periods. Gray circle = cell wall; blue circle = cell membrane; SbtA = bicarbonate transporter; SbtB = PII like signaling, regulator of SbtA and GlgB; GlgB = glycogen branching enzyme.

Fig 7

Cyclic di-AMP is a master regulator of cell volume. Arrows indicate the direction of the effect.