Abstract
The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.
Highlights
Cyclic di-GMP (c-di-GMP)4 has emerged as a central regulator in many environmental and pathogenic bacteria
PA2799 Interacts with the Phosphoreceiver (REC) Domain of the Hybrid Histidine Kinase SagS—We postulated that freestanding PilZ proteins function as c-di-GMP adaptor proteins by binding to their protein targets
The P. aeruginosa PAO1 genomic DNA library used for the bacterial two-hybrid screening was constructed by using the pTRG vector and two engineered pTRG vectors to maximize the number of open reading frames (ORFs) covered by the library
Summary
Cyclic di-GMP (c-di-GMP) has emerged as a central regulator in many environmental and pathogenic bacteria. It is widely hypothesized that free-standing PilZ proteins function as c-diGMP adaptors by binding to their protein partners in a c-diGMP-dependent fashion, experimental evidence supporting this hypothesis is still lacking. This hypothesis was even challenged when a PilZ protein from X. campestris pv. Function of c-di-GMP-binding PilZ Protein sessile life styles and regulates the formation and dispersal of biofilm. The c-di-GMP network of P. aeruginosa PAO1 consists of a large number of DGC and PDE proteins and at least 12 c-di-GMP binding effector proteins. The multi-domain protein Alg contains a c-di-GMP-binding PilZ domain to regulate the biosynthesis of the EPS alginate [10]. The results suggest a novel mechanism used by c-di-GMP to regulate two-component signaling through a PilZ adaptor and establish PA2799 as a new regulatory factor in biofilm formation
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