A Novel C-di-GMP Metabolic Regulatory Pathway Mediated By PdcA/B/C

As a key bacterial second messenger,cyclic di-GMP(c-di-GMP)regulates various physiological processes such as motility,biofilm formation,and virulence.Cellular c-di-GMP levels are regulated by the opposing activities of diguanylate cyclases(DGCs)and c-di-GMPspecific-phosphodiesterases(PDEs).c-di-GMP is synthesized from two GTP by DGCs containing a conserved GGDEF domain,and it is hydrolyzed into linear p Gp G or two GMP by PDEs with an EAL or HD-GYP domain.Bacterial genomes usually encode several c-diGMP-metabolizing proteins.Although the catalytic domains are conserved,many c-di-GMPmetabolizing enzymes have a sensory domain that is predicted to perceive a variety of extracellular signals and intracellular physiological conditions.Intracellular levels of c-diGMP are mainly controlled by modulating the activity of c-di-GMP-metabolizing proteins via the sensory domains adjacent to their enzymatic domains.However,in most cases how c-diGMP-metabolizing enzymes are modulated by their sensory domains remain unclear.In this study,a PAS domain-containing DGC protein PdcA was discovered in Burkholderia thailandensis,revealing the modulation mechanism of Pdc B and PdcC proteins encoded by the same operon on its DGC activity,and elucidated the regulation of this c-di-GMP signaling cascade on motility,biofilm formation and virulence.The main results are as follows:1.According to sequence alignment and domain analysis,six putative DGC proteins,four putative PDE proteins and four proteins with GGDEF/EAL domains were identified in B.thailandensis E264.Sequence alignment showed that the catalytic sites of the c-di-GMPmetabolizing proteins are conserved except for the BTH＿I0751,BTH＿I1393,and the GGDEF domain of BTH＿I2871,indicating that B.thailandensis possesses a complex c-di-GMP regulatory network.2.Swimming motility assays and biofilm formation assays showed that PdcA(BTH＿II2363)regulates motility and biofilm formation,and Cdp A(BTH＿I2871)only affects the motility in B.thailandensis,whereas the others shown no significant changes in motility and biofilm formation,suggesting that PdcA is more important than other c-di-GMPmetabolizing proteins.3.PdcA is composed of an N-terminal PAS domain and a C-terminal GGDEF domain.In vitro enzymatic activity assays and quantification of intracellular c-di-GMP levels confirmed that PdcA possesses DGC activity,which can regulate intracellular c-di-GMP levels in B.thailandensis.Accoring to the structure prediction of PdcA and in vitro enzymatic activity assays and in vivo phenotypic analysis of the truncated PdcA,PAS domain is crucial for the DGC activity of PdcA.4.Analyzing the position of pdc A in the genome,we found that pdc A is located at the end of a gene cluster,and there are two genes pdcB(BTH＿II2364)and pdc C(BTH＿II2364)upstream of it.Co-transcription assays showed that pdc A,pdcB,and pdc C are co-transcribed as an operon.Homologous operon of pdc ABC are widespread in the phylum Proteobacteria.Further phenotypic analysis and the detection of intracellular c-di-GMP levels revealed that Pdc B and PdcC regulate motility and biofilm formation by indirectly modulating intracellular c-di-GMP levels in B.thailandensis.5.Sequence alignment showed that pdc C encodes a single-domain response regulator comprising only the phosphoryl-accepting REC domain,and pdcB encodes a Che C/Che X family phosphatase.Through bacterial two-hybrid assay,GST pull-down assay and in vitro enzymatic activity assays,we found that phosphorylated PdcC inhibits the DGC activity of PdcA by interacting with its PAS domain.Then we found that Pdc B indirectly regulates the DGC activity of PdcA by affecting the phosphorylation level of PdcC,indicating that PdcA,Pdc B,and PdcC constitute a c-di-GMP signaling cascade for cooperatviely regulating the intracellular levels of c-di-GMP in B.thailandensis.6.Through Galleria mellonella(wax moth)larvae infection model and secretion experiments,we found that PdcA,Pdc B,and PdcC modulate the virulence of B.thailandensis by affecting the secretion of T3 SS effectors.In summary,we identified two key c-di-GMP metabolizing proteins,PdcA and Cdp A,in B.thailandensis.The following study showed that PdcA,Pdc B,and PdcC encoded by the same operon constitute a novel c-di-GMP signaling cascade that cooperatviely regulates motility,biofilm formation,and virulence by manipulating the intracellular concentration of c-di-GMP.Our study expands the current knowledge of how bacteria regulate the intracellular c-di-GMP levels.