Azorhizobium caulinodans chemotaxis is controlled by an unusual phosphorelay network

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata . This agriculturally significant symbiotic relationship is important in lowland rice cultivation, and allows for nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA , cheW , cheY2 , cheB , and cheR . Two other chemotaxis genes, cheY1 and cheZ , are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/Receiver pair (termed W2-Rec), follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 or W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1, but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move towards nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over non-specific motion. CheY is an essential mediator of the chemotactic response with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

Predominant phosphorylation patterns in Neisseria meningitidis lipid A determined by top-down MS/MS

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-κB via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS3). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4′-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

ABSTRACT Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3− are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site. IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

Linear 2-Ethylhexyl Imidophosphoric Esters as Effective Rare-Earth Element Extractants

Imidophosphoric organic esters containing phosphoryl groups are potential polydentate ligands and promising extractants of rare-earth elements. For their preparation, a monophosphazene salt [PCl3=N−PCl3]+[PCl6]− and short phosphazene oligomers of the general formula [Cl–(PCl2=N)n–PCl3]+[PCl6]−, where n = 4–7, were synthesized via living cationic polymerization of Cl3P=NSiMe3 and used as starting compounds. All phosphazenes were reacted with 2-ethylhexanol to obtain the corresponding esters of imidophosphoric acids (EIPAs). The formation of imidophosphoric acids occurs due to the phosphazene-phosphazane rearrangement of –P(OR)2=N– or –P(OH)(OR)=N– units, where R = 2-ethylhexyl. The prepared EIPAs were characterized by 1H, 31P NMR, and MALDI-TOF analyses and their extractive capacity towards lanthanide ions in aqueous solutions of nitric acid was examined. The EIPAs are mixtures of mono-, di-, and trifunctional compounds of the type HxA, where x = 1–3, which can form chelate complexes of lanthanide ions [Ln(A)z], where z = 3–6, depending on the chain length. The longer chain EIPAs are more suitable for collective rare-earth elements extraction. A comparison of the extraction properties of the EIPAs with the industrially used polyalkylphosphonitrilic acid (PAPNA) was drawn.

STRUCTURAL AND SPECTRAL STUDIES OF THE MIXED-LIGAND CATION COMPLEX OF LANTHANUM [Lа(L)2bipy2]BPh4 WITH CARBACYLAMIDOPHOSPHATE (CAPH) TYPE LIGAND AND 2,2'-BIPYRIDINE

A new cationic mixed-ligand complex [Lа(L)2bipy2]BPh4 (where L-= bis(N,N'-diethylamide)(N'-trichloroacetyl)-triamidophosphate anion, bipy = 2,2'-bipyridine) has been synthesized and studied by the means of IR, 1H NMR spectroscopy, thermogravimetric and X-ray structural analyses. Low-frequency shifts of the absorption bands of the carbonyl and phosphoryl groups of phosphorylated ligand in the IR spectra of the complex compared with similar absorption bands in the spectrum of "free" CAPh ligand are Δν(C = O) = 117 cm–1 and Δν(P = O) = 137 cm–1. The analysis of integral signal intensity in the investigated NMR spectra of coordination compounds [Lа(L)2bipy2]BPh4 indicates the molar ratio of ligand : bipyridine : tetraphenylborate anion as a 2:2:1, which corresponds to the proposed structure of the complexes. The compound has been obtained in monocrystalline form. The structure of the complex has been determined by X-ray structural analysis, its ionic structure was proved, and the coordination of two CAPh ligands through the oxygen atoms of the carbonyl and phosphoryl groups was confirmed. Based on the structural data, it was determined that the La3+ ion is octocoordinated (surrounded by four oxygen atoms from two chelated phosphoryl ligands and four nitrogen atoms from two 2,2'-bipyridine molecules). The coordination polyhedron of central ion is interpreted as a square antiprism. Complex cations and tetraphenylborate anions are connected both by electrostatic interaction and by weak intermolecular C – H ∙∙∙ π-contacts between phenyl substituents of BPh4- and molecules of 2,2'-bipyridine. It was established by thermogravimetric analysis that the complex [Lа(L)2bipy2]BPh4 obtained is thermally stable up to a temperature of 150 °C. Significant decomposition of the complex begins at a temperature of 150 °C, occurs in one stage and most intensively continues up to 300 °C. The total weight loss is 78 %.

CoralP: Flexible visualization of the human phosphatome

Protein phosphatases and kinases play critical roles in a host of biological processes and diseases via the removal and addition of phosphoryl groups. While kinases have been extensively studied for decades, recent findings regarding the specificity and activities of phosphatases have generated an increased interest in targeting phosphatases for pharmaceutical development. This increased focus has created a need for methods to visualize this important class of proteins within the context of the entire phosphatase protein family. Here, we present CoralP, an interactive web application for the generation of customizable, publication-quality representations of human phosphatome data. Phosphatase attributes can be encoded through edge colors, node colors, and node sizes. CoralP is the first and currently the only tool designed for phosphatome visualization and should be of great use to the signaling community. The source code and web application are available at https://github.com/PhanstielLab/coralp and http://phanstiel-lab.med.unc.edu/coralp respectively.

Dinuclear zinc synergistic catalytic asymmetric phospha-Michael/Michael cascade reaction: synthesis of 1,2,3-trisubstituted indanes bearing phosphoryl groups

A series of new 1,2,3-trisubstituted indane derivatives containing phosphorus groups are efficiently obtained via asymmetric dinuclear zinc synergistic catalytic phospha-Michael/Michael cascade reaction.