Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis

Abstract

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5′-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

The c‐di‐AMP signaling system influences stress tolerance and biofilm formation of Streptococcus mitis

“…To analyze a potential effect on growth rate in more detail, we performed growth experiments in the presence of sub‐lethal concentrations of antibacterial compounds and compared it to the growth in the absence of antibacterial supplements. In TSB without any supplementation, the WT and Δ cdaA and Δ pde1 mutants grew relatively similarly, but the Δ cdaA mutant generally reached a slightly lower OD in the stationary phase (Figure 2a, b and Rørvik et al, 2020 ). The Δ pde2 mutant on the other hand displayed a reduced growth rate and reached a lower OD in the stationary phase than the other strains (Figure 2c and Rørvik et al, 2020 ).…”

“…In TSB without any supplementation, the WT and Δ cdaA and Δ pde1 mutants grew relatively similarly, but the Δ cdaA mutant generally reached a slightly lower OD in the stationary phase (Figure 2a, b and Rørvik et al, 2020 ). The Δ pde2 mutant on the other hand displayed a reduced growth rate and reached a lower OD in the stationary phase than the other strains (Figure 2c and Rørvik et al, 2020 ). In TSB supplemented with ampicillin (0.0625 mg/L), the Δ pde1 mutant grew slightly faster than the wild type in the exponential growth phase compared to the WT, while the Δ pde2 and the Δ cdaA mutant grew slower compared to the WT (Figure 2d–f ).…”

“…The c‐di‐AMP signaling system is involved in regulating the growth of S. mitis in unmodified TSB at 37°C in shaking culture (Figure 2a‐c and (Rørvik et al, 2020 )). Growth was also analyzed under static growth conditions.…”

“…The bacterial strains used in this study are summarized in Table 1 . The deletion mutants and knock‐back (KB) strains had been generated by markerless gene editing in a previous study (Rørvik et al, 2020 ). Bacteria were, unless otherwise stated, grown in tryptone soya broth (TSB) (Oxoid) at 37°C in a humidified atmosphere containing 5% CO 2 .…”

“…In addition, c‐di‐AMP is recognized by several receptors of the innate immune system, leading to type I interferon production or NF‐κB activation (Barker et al, 2013 ; McFarland et al, 2017 ; Parvatiyar et al, 2012 ; Woodward et al, 2010 ; Xia et al, 2018 ), and it is therefore directly involved in crosstalk between the microbe and the host (Andrade et al, 2016 ; Woodward et al, 2010 ). We have previously identified and characterized one c‐di‐AMP‐producing diadenylate cyclase (CdaA) and two c‐di‐AMP‐degrading phosphodiesterases (Pde1 and Pde2) in S. mitis CCUG 31611 (Rørvik et al, 2020 ) and showed that the c‐di‐AMP signaling system regulates growth, metabolism, colony morphology, and chain length in S. mitis .…”