RESUMEN
Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.
Asunto(s)
Cronobacter sakazakii , Cronobacter , Lactante , Humanos , Cronobacter sakazakii/genética , Huésped Inmunocomprometido , SacarosaRESUMEN
Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in Pseudomonas aeruginosa. Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Biopelículas/crecimiento & desarrollo , Catálisis , Fosfatos de Dinucleósidos/química , Activación Enzimática , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/genética , Unión Proteica , Conformación Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Bacterial cyclic-di-GMP (c-di-GMP) production is associated with biofilm development and the switch from acute to chronic infections. In Pseudomonas aeruginosa, the diguanylate cyclase (DGC) SiaD and phosphatase SiaA, which are co-transcribed as part of a siaABCD operon, are essential for cellular aggregation. However, the detailed functions of this operon and the relationships among its constituent genes are unknown. Here, we demonstrate that the siaABCD operon encodes for a signaling network that regulates SiaD enzymatic activity to control biofilm and aggregates formation. Through protein-protein interaction, SiaC promotes SiaD diguanylate cyclase activity. Biochemical and structural data revealed that SiaB is an unusual protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD and regulate c-di-GMP levels and subsequent virulence phenotypes. Collectively, our data provide insights into the molecular mechanisms underlying the modulation of DGC activity associated with chronic infections, which may facilitate the development of antimicrobial drugs.
