YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity

Abstract

The cyclic dinucleotide c-di-AMP [corrected] synthesized by the diadenylate cyclase domain was discovered recently [corrected] as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5'-pApA and 5'-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K(m)) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the DeltayybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.

FIGURE 1.

Domain architecture of YybT family proteins (COG3387) with the three protein constructs studied in this work indicated.

FIGURE 2.

Specific phosphodiesterase activity of YybT_84–659. A, partial alignment of DHH-DHHA1 domain sequences with the conserved residues for metal ion coordination highlighted. B, enzymatic activity of YybT against nonphysiological p-nitrophenol substrates. C, structural model of DHH_YybT active site with the metal ions shown as balls and the coordinating residues as sticks.

FIGURE 3.

Degradation of c-di-AMP by YybT. A, HPLC analysis of the hydrolysis of c-di-AMP by YybT_84–659. The assay conditions are described under “Experimental Procedures”. B, steady-state kinetic analysis of the hydrolysis of c-di-AMP catalyzed by YybT_84–659.

FIGURE 4.

Metal and pH dependence of c-di-AMP degradation by YybT_84–659. A, metal dependence of YybT-catalyzed c-di-AMP hydrolysis. The enzymatic assay conditions are described under “Experimental Procedures.” B, Mn²⁺ and Mg²⁺ concentration dependence of c-di-AMP hydrolysis. C, pH dependence of DHH_YybT domain activity.

FIGURE 5.

ATPase activity of the GGDEF_YybT domain. A, HPLC analysis of ATP hydrolysis catalyzed by YybT_84–303. B, steady-state kinetic measurement of ATPase domain activity in the presence and absence of nonhydrolyzable ATP analog. C, relative ATPase activity of YybT_84–303 and its mutants in the presence of Mg²⁺ and Mn²⁺. D, sequence comparison of the GGDEF_YybT domain with some characterized GGDEF domains. The three residues mutated in this study are indicated by the vertical arrows, whereas the residues for guanosine binding in orthodox GGDEF domains are indicated by asterisks. GdpP (S. aureus) is a homolog of YybT. The GGDEF domain of GdpS (S. aureus) exhibits residual GTPase activity. The GGDEF domains of PleD (C. crescentus) (30), WspR (P. aeruginosa) (54), and AxDGC2 (Acetobacter xylinum) function as diguanylate cycles (55). The GGDEF domain of Cc3396 binds GTP as a regulatory domain.

FIGURE 6.

Inhibition of the phosphodiesterase activity by ppGpp. A, ppGpp concentration dependence of the c-di-AMP hydrolyzing activity. The IC₅₀ value is determined from the midpoint of the plot. B, double-reciprocal plots of c-di-AMP hydrolyzing activity at various ppGpp concentrations. The experimental conditions are described under “Experimental Procedures.”

FIGURE 7.

Acid and DNA damage resistance. A, relative survival of wild type and ΔyybT strain under acid stress conditions. B, spore formation efficiency for the wild type B. subtilis and ΔyybT mutant in the presence of DNA damage-inducing agent nalidixic acid. The spores (%) were obtained as described under “Experimental Procedures.”