RESUMO
Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Proteínas de Bactérias/química , Histidina Quinase/química , Pseudomonas aeruginosa/metabolismo , VirulênciaRESUMO
The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesium-based hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established.
Assuntos
Proteínas de Bactérias/fisiologia , Ritmo Circadiano/fisiologia , Cianobactérias/fisiologia , Magnésio/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cianobactérias/metabolismo , Simulação de Dinâmica Molecular , FosforilaçãoRESUMO
In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor-P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5-receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW-receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.
Assuntos
Proteínas de Escherichia coli/química , Histidina Quinase/química , Proteínas Quimiotáticas Aceptoras de Metil/química , Transdução de Sinais , Quimiotaxia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Modelos Moleculares , MutaçãoRESUMO
Recognition of the host plant is a prerequisite for infection by pathogenic bacteria. However, how bacterial cells sense plant-derived stimuli, especially chemicals that function in regulating plant development, remains completely unknown. Here, we have identified a membrane-bound histidine kinase of the phytopathogenic bacterium Xanthomonas campestris, PcrK, as a bacterial receptor that specifically detects the plant cytokinin 2-isopentenyladenine (2iP). 2iP binds to the extracytoplasmic region of PcrK to decrease its autokinase activity. Through a four-step phosphorelay, 2iP stimulation decreased the phosphorylation level of PcrR, the cognate response regulator of PcrK, to activate the phosphodiesterase activity of PcrR in degrading the second messenger 3',5'-cyclic diguanylic acid. 2iP perception by the PcrK-PcrR remarkably improves bacterial tolerance to oxidative stress by regulating the transcription of 56 genes, including the virulence-associated TonB-dependent receptor gene ctrA. Our results reveal an evolutionarily conserved, inter-kingdom signaling by which phytopathogenic bacteria intercept a plant hormone signal to promote adaptation to oxidative stress.
Assuntos
Citocininas/metabolismo , Histidina Quinase/metabolismo , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Histidina Quinase/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMO
Agrobacterium tumefaciens, a soil-born phytopathogenic bacterium, is well known as a nature's engineer due to its ability to genetically transform the host by transferring a DNA fragment (called T-DNA) from its Ti plasmid to host-cell genome. To combat the harsh soil environment and seek the appropriate host, A. tumefaciens can sense and be attracted by a large number of chemical compounds released by wounded host. As a member of α-proteobacterium, A. tumefaciens has a chemotaxis system different from that found in Escherichia coli, since many chemoattractants for A. tumefaciens chemotaxis are virulence (vir) inducers. However, advances in the study of the chemotaxis paradigm, E. coli chemotaxis system, have provided enough information to analyze the A. tumefaciens chemotaxis. At low concentration, chemoattractants elicit A. tumefaciens chemotaxis and attract the species to the wound sites of the host. At high concentration, chemoattractants induce the expression of virulence genes and trigger T-DNA transfer. Recent studies on the VirA and ChvE of the vir-induction system provide some evidences to support the crosstalk between chemotaxis and vir-induction. This review compares the core components of chemotaxis signaling system of A. tumefaciens with those observed in other species, discusses the connection between chemotaxis and vir-induction in A. tumefaciens, and proposes a model depicting the signaling crosstalk between chemotaxis and vir-induction.