Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.
- MeSH
- adenin analogy a deriváty farmakologie MeSH
- Arabidopsis cytologie účinky léků genetika metabolismus MeSH
- cytokininy metabolismus MeSH
- extracelulární tekutina metabolismus MeSH
- geneticky modifikované rostliny MeSH
- histidinkinasa genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- signální transdukce MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains.
- MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu * MeSH
- fosforylace MeSH
- histidinkinasa chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- rozpustnost MeSH
- rychlé screeningové testy * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- DNA bakterií genetika MeSH
- fenotyp MeSH
- genom bakteriální genetika MeSH
- histidinkinasa genetika MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- rezistence na vankomycin genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus haemolyticus účinky léků genetika izolace a purifikace MeSH
- teikoplanin farmakologie MeSH
- vankomycin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
The phytohormone cytokinin is a regulator of numerous processes in plants. In Arabidopsis (Arabidopsis thaliana), the cytokinin signal is perceived by three membrane-located receptors named ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and AHK4/CRE1. How the signal is transmitted across the membrane is an entirely unknown process. The three receptors have been shown to operate mostly in a redundant fashion, and very few specific roles have been attributed to single receptors. Using a forward genetic approach, we isolated constitutively active gain-of-function variants of the AHK2 and AHK3 genes, named repressor of cytokinin deficiency2 (rock2) and rock3, respectively. It is hypothesized that the structural changes caused by these mutations in the sensory and adjacent transmembrane domains emulate the structural changes caused by cytokinin binding, resulting in domain motion propagating the signal across the membrane. Detailed analysis of lines carrying rock2 and rock3 alleles revealed how plants respond to locally enhanced cytokinin signaling. Early flowering time, a prolonged reproductive growth phase, and, thereby, increased seed yield suggest that cytokinin regulates various aspects of reproductive growth. In particular, it counteracts the global proliferative arrest, a correlative inhibition of maternal growth by seeds, an as yet unknown activity of the hormone.
- MeSH
- Arabidopsis anatomie a histologie genetika fyziologie MeSH
- časové faktory MeSH
- cytokininy metabolismus farmakologie MeSH
- geneticky modifikované rostliny MeSH
- histidinkinasa genetika MeSH
- květy genetika fyziologie ultrastruktura MeSH
- meristém genetika fyziologie ultrastruktura MeSH
- mikroskopie elektronová rastrovací MeSH
- mutace * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny huseníčku genetika MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus farmakologie MeSH
- rozmnožování genetika fyziologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- signální transdukce účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.
- MeSH
- biokatalýza účinky léků MeSH
- fosforylace účinky léků MeSH
- hem chemie metabolismus MeSH
- histidinkinasa chemie genetika metabolismus MeSH
- kinetika MeSH
- kyslík chemie metabolismus MeSH
- molekulární struktura MeSH
- mutageneze cílená MeSH
- Myxococcales enzymologie MeSH
- sulfan chemie farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.
- MeSH
- Aeromonas salmonicida genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fosforylace MeSH
- hem chemie metabolismus MeSH
- histidin chemie metabolismus MeSH
- histidinkinasa chemie genetika metabolismus MeSH
- klonování DNA MeSH
- kyselina aspartová chemie metabolismus MeSH
- kyslík chemie metabolismus MeSH
- Myxococcales chemie enzymologie MeSH
- proteinové domény MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- signální transdukce * MeSH
- strukturní homologie proteinů MeSH
- vodík-deuteriová výměna MeSH
- železo chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
