A c-di-GMP Signaling Cascade Controls Motility, Biofilm Formation, and Virulence in Burkholderia thailandensis.

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 phosphodiesterases (PDEs). Beyond that, the enzymatic activities of c-di-GMP metabolizing proteins are controlled by a variety of extracellular signals and intracellular physiological conditions. Here, we report that pdcA (BTH_II2363), pdcB (BTH_II2364), and pdcC (BTH_II2365) are cotranscribed in the same operon and are involved in a regulatory cascade controlling the cellular level of c-di-GMP in Burkholderia thailandensis. The GGDEF domain-containing protein PdcA was found to be a DGC that modulates biofilm formation, motility, and virulence in B. thailandensis. Moreover, the DGC activity of PdcA was inhibited by phosphorylated PdcC, a single-domain response regulator composed of only the phosphoryl-accepting REC domain. The phosphatase PdcB affects the function of PdcA by dephosphorylating PdcC. The observation that homologous operons of pdcABC are widespread among betaproteobacteria and gammaproteobacteria suggests a general mechanism by which the intracellular concentration of c-di-GMP is modulated to coordinate bacterial behavior and virulence. IMPORTANCE The transition from planktonic cells to biofilm cells is a successful strategy adopted by bacteria to survive in diverse environments, while the second messenger c-di-GMP plays an important role in this process. Cellular c-di-GMP levels 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-di-GMP-metabolizing enzymes are modulated by their sensory domains remains unclear. Here, we reveal a new c-di-GMP signaling cascade that regulates motility, biofilm formation, and virulence in B. thailandensis. While pdcA, pdcB, and pdcC constitute an operon, the phosphorylated PdcC binds the PAS sensory domain of PdcA to inhibit its DGC activity, with PdcB dephosphorylating PdcC to derepress the activity of PdcA. We also show this c-di-GMP regulatory model is widespread in the phylum Proteobacteria. Our study expands the current knowledge of how bacteria regulate intracellular c-di-GMP levels.

HpaR, the Repressor of Aromatic Compound Metabolism, Positively Regulates the Expression of T6SS4 to Resist Oxidative Stress in Yersinia pseudotuberculosis.

HpaR, a MarR family transcriptional regulator, was first identified in Escherichia coli W for its regulation of the hpa-meta operon. Little else is known regarding its functionality. Here, we report that in Yersinia pseudotuberculosis, HpaR negatively regulates the hpa-meta operon similar to in E. coli W. To investigate additional functions of HpaR, RNA sequencing was performed for both the wild-type and the ΔhpaR mutant, which revealed that the type VI secretion system (T6SS) was positively regulated by HpaR. T6SS4 is important for bacteria resisting environmental stress, especially oxidative stress. We demonstrate that HpaR facilitates bacteria resist oxidative stress by upregulating the expression of T6SS4 in Y. pseudotuberculosis. HpaR is also involved in biofilm formation, antibiotic resistance, adhesion to eukaryotic cells, and virulence in mice. These results greatly expand our knowledge of the functionality of HpaR and reveal a new pathway that regulates T6SS4.