Direct observation of electrogenic NH4+ transport in ammonium transport (Amt) proteins

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH₄⁺ scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH₄⁺/NH₃ transport is used instead in acid–base and pH homeostasis in kidney or NH₄⁺/NH₃ (and eventually CO₂) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.Ammonium transport (Amt) proteins are a class of trimeric, integral membrane proteins found throughout all domains of life. Despite moderate primary sequence homologies, distinct family members from bacteria, archaea, and eukarya (including humans) share conserved structural features and a high number of conserved amino acid residues that are considered functionally relevant (1–4). Although the involvement of all Amt proteins in transporting NH₄⁺/NH₃ across biological membranes is undisputed, their functional context is diverse. Prokaryotes and plants use Amt proteins to scavenge NH₄⁺/NH₃—a preferred nitrogen source for cell growth—from their environment, whereas mammals use Amt orthologs, the Rhesus proteins, for detoxification and ion homeostasis in erythrocytes and in the kidney and liver tissues (1, 5, 6).Three decades ago, Kleiner and coworkers suggested that Amt proteins are secondary active and electrogenic transporters for ammonium (7–9). Various groups have subsequently confirmed this finding by two-electrode voltage-clamp experiments with protein produced recombinantly from RNA injected into Xenopus laevis oocytes. Here, plant Amt and Rhesus proteins were the main object of study, but some mechanistic details remained unclear, in particular the distinction between electrogenic NH₄⁺ uniport (10–13), NH₃/H⁺ symport (11, 12), or electroneutral NH₄⁺/H⁺ antiport (14, 15). In contrast, bacterial Amt proteins were described as passive channels for the uncharged gas ammonia (NH₃) (16). The first crystal structure for an Amt family member, AmtB from Escherichia coli (17), was interpreted to support this hypothesis, and an ongoing controversy concerning the transported species has persisted in the field ever since. Several points have been raised to challenge the possibility of gas channeling, the most critical of which seems to be that at physiological pH the protonation equilibrium of NH₃—with a pK_a of 9.4—would be >99% on the side of charged NH₄⁺. This point implies that the import of neutral ammonia gas must be preceded by extracellular deprotonation and followed immediately by intracellular protonation. In summary, the import of NH₃ would thus result in a net NH₄⁺/H⁺ antiport. Such a mechanism would be electroneutral, but it would be secondary active in the presence of a proton motive force, resulting in a vectorial pumping of ammonium out of the cell—which is, of course, physiologically unreasonable. A second point is that biological membranes are themselves highly permeable for uncharged ammonia, with a permeability coefficient, P_d = 10⁻³ cm·s⁻¹, similar to that of water (18), such that a dedicated transport protein would hardly be required. Westerhoff and coworkers have argued that active Amt transport thus is imperative and that cells must be able to quickly block Amt transport upon intracellular accumulation of ammonium to avoid uncoupling of the proton gradient through back-diffusion of NH₃ (19). In prokaryotes and some plants, this blocking is the task of regulatory GlnK proteins belonging to the signal transducing P_II family that bind to corresponding ammonium transporters when their regulatory ligand 2-oxoglutarate, the primary metabolic acceptor for NH₄⁺ during nitrogen assimilation, is depleted (20).The high expectations to understand the mechanism of Amt transport from 3D structures have not been met to date. The available structures of E. coli AmtB (17, 21) and its complex with GlnK (22, 23) of A. fulgidus Amt-1 (24), Nitrosomonas europaea Rh50 (25, 26), and human RhCG (27) all show the same, inward-facing state of the protein. Such apparent structural rigidity would match the picture of a fast channel, whereas active transport is generally considered to involve conformational changes that expose a binding site for the cargo molecule(s) alternatingly to either side of the membrane (28). In addition, the difficulties to detect NH₄⁺/NH₃ and to assay Amt transport led to a lack of functional studies carried out in vitro on well-defined systems. An uptake assay with AmtB reconstituted in proteoliposomes was described to provide evidence for passive gas channeling (17), but the methodology was later contested (2). Assays based on the detection of radioactive methylammonium (MA) uptake were only carried out in whole cells of E. coli, and studies with voltage-clamp electrophysiology using Amt-1 reconstituted in planar lipid bilayers did not yield conclusive results (our work). A series of potentially important variants have been produced (29–39), but the lack of an adequate functional assay has precluded definite conclusions.The debate concerning the transport mechanism of Amt proteins has not been settled to date, necessitating a reliable functional in vitro assay. The finding that electrogenic transport was observed in X. laevis oocytes, but not in the far smaller membrane patch of a planar lipid bilayer setup, suggested that the transport rate of Amt proteins was possibly too low to lead to a detectable current response, unless a larger number of protein units were incorporated into the bilayer. We have therefore focused on a controlled method of in vitro electrophysiology that allows the simultaneous activation of >10⁸ protein units, the solid-supported membrane (SSM) electrophysiology (40). With this approach, pioneered by Fendler and coworkers, we were able to detect robust ion currents from isolated and reconstituted Amt proteins.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.Living organisms have developed dynamic strategies to explore nutrients in the environment. Morphological plasticity of plant roots and microorganisms is often compared with foraging behavior of animals. Plant roots are highly dynamic systems because they can modify their structure to reach nutrient resources in soil and optimize their nutrient uptake capacities. This strategy appears to be associated with morphological adaptation, because plants are sessile in nature and nutrient availabilities in soil are often altered by surrounding biotic and abiotic factors and climate changes. Morphological modifications of plant root systems are particularly prominent when they grow in soil environments with unbalanced nutrient availabilities (1–4). Among the essential elements required for plant growth, nitrogen (N) has a particularly strong effect on root development (1–6). Lateral roots can be developed in N-rich soil patches where adequate amounts of nitrate (NO₃⁻) or ammonium (NH₄⁺) are available, whereas this local outgrowth of lateral roots is restricted in N-deficient patches (7–9). In addition to these local N responses, lateral root growth is stimulated in response to mild N deficiency and suppressed under excess N supply by systemic plant signals carrying information on the nutritional status of distant plant organs (4, 10–13). These morphological responses are important for plant fitness and N acquisition, despite the cost for structuring the root system architecture (2, 6). However, lateral root growth is not sustained when plants are deprived of N for an extended period (4). Under such severe circumstances, the development of new lateral roots should rather be restricted to prevent the risk of extending roots into N-poor environments. Economizing the cost for root development appears to be an important morphological strategy for plant survival.To modify root traits in response to changing N availabilities, plants use various types of signaling molecules including hormones and small RNAs (10, 13–17). In particular, auxin signaling proteins and auxin transporters have been proven essential for lateral root development in response to local nitrate supplies (10, 14–17). These proteins are involved in increasing auxin sensitivity or auxin accumulation at lateral root initials or lateral root tips exposed to NO₃⁻, and the NRT1.1 nitrate transporter has been suggested to play a key role in NO₃⁻ sensing (8, 17, 18). In addition, mutations of the nitrate transporter NRT2.1 have been shown to repress or stimulate lateral root initiation depending on N conditions and sucrose supply (12, 19). Thus, N-dependent root development is apparently under control of complex mechanisms, although its signaling components have remained largely unidentified. In this study, we have identified several homologs of the CLE (CLAVATA3/ESR-related) gene family (20–24) to be up-regulated by N deficiency and involved in this yet unresolved regulatory mechanism. CLAVATA3 (CLV3) is known as a signaling peptide that binds to the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK) and controls stem cell differentiation in the shoot apical meristem (25–32). CLE-receptor signaling modules are also known to control meristem function in the primary and lateral roots (33–35). The N-responsive CLE peptides described in the present study belong to the group of CLE peptides with the highest sequence similarity to CLAVATA3 (CLV3) (21–23) and may partly substitute for CLV3 in the shoot apical meristem (31, 36, 37). Our present findings indicate that the N-responsive CLE peptides and CLV1 are signaling components required for translating an N-deficient nutritional status into a morphological response inhibiting the outgrowth of lateral root primordia in Arabidopsis. The present study demonstrates a unique function of the CLE-CLV1 signaling module in roots and provides new insights into signaling mechanisms regulating the expansion of the plant root system in N-deficient environments.     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Slow hydrogen-bond switching dynamics at the water surface revealed by theoretical two-dimensional sum-frequency spectroscopy

Using our newly developed explicit three-body (E3B) water model, we simulate the surface of liquid water. We find that the timescale for hydrogen-bond switching dynamics at the surface is about three times slower than that in the bulk. In contrast, with this model rotational dynamics are slightly faster at the surface than in the bulk. We consider vibrational two-dimensional (2D) sum-frequency generation (2DSFG) spectroscopy as a technique for observing hydrogen-bond rearrangement dynamics at the water surface. We calculate the nonlinear susceptibility for this spectroscopy for two different polarization conditions, and in each case we see the appearance of cross-peaks on the timescale of a few picoseconds, signaling hydrogen-bond rearrangement on this timescale. We thus conclude that this 2D spectroscopy will be an excellent experimental technique for observing slow hydrogen-bond switching dynamics at the water surface.Interfaces play important roles in many disciplines of science. The water liquid/vapor interface, for example, is of great interest in chemistry, biology, and earth science and is an important model system for water in a heterogeneous environment. Of particular interest is understanding the extent to which the structure and dynamics, and ultimately reactivity, of water at the interface differ from those in the bulk. For example, how does the distribution of hydrogen bonds differ between interfacial and bulk water? How anisotropic is the orientation of the water molecules at the interface? In terms of dynamics, how do the diffusion constant, rotational relaxation time, and hydrogen-bond rearrangement time vary as the interface is approached? One can also consider vibrational dynamics processes such as energy relaxation and transfer.One important technique for addressing these questions is computer simulation. Models used in these calculations for the water surface range from rigid, fixed-point-charge two-body models (1–3), to fluctuating charge or polarizable models (4, 5), to ab initio molecular dynamics calculations (6–10). Regarding static properties, for example, some effort has been expended toward understanding what fraction of H atoms in the surface layer are hydrogen bonded, and what fraction of molecules do not donate any hydrogen bonds (nondonors or “acceptor-only” molecules) (6, 9). In terms of dynamics, it is generally found that diffusion is faster at the interface than in the bulk (1, 4, 10), and rotational relaxation is also faster (3, 6, 7, 10). On the other hand, two studies with fixed-charge two-body models show that hydrogen-bond rearrangement is slower at the interface (2, 3), whereas one study with a fluctuating-charge model shows that hydrogen-bond rearrangement is faster (5). In this latter study the authors conclude that this is generally true for polarizable models.Because of its surface sensitivity, vibrational sum-frequency generation (SFG) spectroscopy (11, 12) has become one of the most powerful experimental techniques for the study of interfaces, including the one separating liquid water and its vapor (13–28). In a vibrational SFG experiment, infrared (IR) and visible laser pulses are incident on the interface, and the signal is detected at the sum of the frequencies of these incoming beams. For the water liquid/vapor interface one can think of the SFG intensity as the vibrational spectrum of the water molecules near the surface (29, 30). Intensity-level SFG spectra of the OH stretching mode of water show two major features for this system. A sharp peak near 3,700 cm⁻¹ indicates the existence of dangling or “free” OH groups at the water surface. The other broad band in the frequency region from 3,000 to 3,600 cm⁻¹ is interpreted as arising from hydrogen-bonded OHs (13, 14).Further interpretation of SFG results was catalyzed by two major advances. First, studying the isotopically dilute HOD in D₂O (or H₂O) system has helped in the interpretation of spectra, because the frequency mismatch of OH and OD stretches largely eliminates the effects of vibrational couplings, which greatly complicate the measured spectra for neat water (31, 32). Second, the invention of phase-sensitive SFG enables the direct measurement of the imaginary part of the second-order complex susceptibility χ₂ (whereas the conventional experiments measure |χ₂|²) (33, 34). Because Im(χ₂) contains only resonant contributions, it is analogous to an absorption spectrum and is easier to interpret and to calculate. Moreover, Im(χ₂) is signed, and the sign is related to the projection of the OH (OD) transition dipole onto the surface normal. Recently, Shen and coworkers measured Im(χ₂) for both the neat and the isotope-labeled water liquid/vapor interfaces (35, 36). These phase-sensitive SFG results for HOD/D₂O show three major features: a sharp positive peak at about 3,700 cm⁻¹ corresponding to the upward-pointing dangling OH bonds and negative and positive bands at about 3,500 and 3,300 cm⁻¹, respectively, attributed to hydrogen-bonded water OHs (36). The latter two peaks were interpreted by the authors as “water-like” molecules with downward-pointing OH bonds and “ice-like” molecules with upward-pointing OH bonds, respectively (36, 37).As mentioned above, phase-sensitive SFG allows for a better comparison between experimental results and theoretical calculations. Although recent papers by Geissler, Shen, and coworkers show that in principle magnetic-dipole and electric-quadrupole terms should be included in a correct SFG calculation (38–40), in practice nearly all calculations have used the electric-dipole approximation. Most of the widely used two-body water models fail to reproduce the positive band in the low-frequency region of the phase-sensitive SFG spectrum (41–46). Morita and coworkers have developed a polarizable and flexible classical water model that successfully qualitatively reproduces Im(χ₂) of the neat H₂O surface, and they assigned the positive signal in the hydrogen-bonding region to induced dipoles perpendicular to the water surface (28, 47, 48). This conclusion was also in agreement with their results from hybrid quantum mechanics/molecular mechanics molecular dynamics (MD) simulations (49). Our group has used the newly developed explicit three-body (E3B) water model (50, 51), which includes three-molecule interactions, to calculate Im(χ₂) using a mixed quantum/classical approach. The calculations also qualitatively reproduce the experimental spectra. The two features in the hydrogen-bonding region were found to result from canceling contributions from water molecules with different hydrogen-bonding configurations (46, 52), with especially large contributions from four-hydrogen–bonded double-donor molecules and two-hydrogen–bonded single-donor molecules.Although conventional SFG spectroscopy can provide structural information about an interface, in the case of water, where the spectrum is dominated by inhomogeneous broadening, it is unable to probe the dynamics. To study dynamics, one-dimensional (1D)SFG needs to be extended to two dimensions (2D), much like IR spectroscopy has recently been extended to 2DIR (53). In a time-domain 2DIR experiment, the sample is subjected to three IR pulses, separated by two time intervals t₁ and t₂, and the signal is heterodyne detected at a time t₃ later. The signal is then Fourier transformed in t₁ and t₃, leading to two frequency dimensions ω₁ and ω₃. A series of 2DIR spectra is collected as a function of t₂, the “waiting time.” Roughly speaking, the 2DIR spectrum can be thought of as the joint probability density that the chromophore has frequency ω₁ at time 0 and ω₃ at time t₂. Thus, the experiment naturally reports on dynamic processes such as spectral diffusion and chemical exchange (53). 2DIR spectroscopy has been widely used to study dynamics in bulk water, including hydrogen-bond dynamics (54–60), rotations (61–63), and vibrational energy transfer (64–69). However, 2DIR, a third-order nonlinear spectroscopy, is not surface sensitive. Therefore, 2DSFG, a fourth-order nonlinear spectroscopy, is needed to study the dynamics at the interfacial region.In 2DSFG, four laser beams are incident on the sample: three time-delayed IR pulses followed by a visible pulse (70). Although some related and beautiful ultrafast pump-probe SFG and homodyne-detected 2DSFG experiments have been performed to measure vibrational relaxation, rotational dynamics, and vibrational energy transfer at the water surface (71–76), to the best of our knowledge the heterodyne-detected 2DSFG experiment for the air/water interface (and especially desirably, for HOD/D₂O or HOD/H₂O) has not yet been performed. Indeed, only two heterodyne-detected 2DSFG experiments on any system have been reported (70, 77). Xiong et al. measured the heterodyne-detected 2DSFG spectrum of CO molecules adsorbed on a platinum surface (70), whereas Singh et al. measured the heterodyne-detected 2DSFG spectrum for the interface of HOD/D₂O with the positively charged surfactant cetyltrimethylammonium bromide (77). This latter system provides a stronger signal than that for the air/water interface, because the electric field from the surfactant produces substantial alignment of the water molecules. A theoretical 2DSFG calculation by Nagata et al. using a classical approach has appeared for water at a lipid monolayer interface (78).In this paper, we simulate the liquid/vapor interface using our E3B water model (51, 52). We calculate the hydrogen-bond rearrangement time-correlation function for the surface molecules, finding that it decays on a timescale of about 4 ps, which is significantly slower than the corresponding timescale for the bulk. We can contrast this to the timescale for rotational motion, which for our model is faster at the surface than in the bulk. We also calculate the 2DSFG signal for different polarizations, as a function of waiting time. Cross-peaks grow in on the timescale of the hydrogen-bond rearrangment time. This demonstrates the promise of the 2DSFG technique for an experimental measurement of spectral diffusion and chemical exchange at the water surface and hence an experimental measurement of structural relaxation in the interfacial region.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea

Archaebacterial halophiles (Haloarchaea) are oxygen-respiring heterotrophs that derive from methanogens—strictly anaerobic, hydrogen-dependent autotrophs. Haloarchaeal genomes are known to have acquired, via lateral gene transfer (LGT), several genes from eubacteria, but it is yet unknown how many genes the Haloarchaea acquired in total and, more importantly, whether independent haloarchaeal lineages acquired their genes in parallel, or as a single acquisition at the origin of the group. Here we have studied 10 haloarchaeal and 1,143 reference genomes and have identified 1,089 haloarchaeal gene families that were acquired by a methanogenic recipient from eubacteria. The data suggest that these genes were acquired in the haloarchaeal common ancestor, not in parallel in independent haloarchaeal lineages, nor in the common ancestor of haloarchaeans and methanosarcinales. The 1,089 acquisitions include genes for catabolic carbon metabolism, membrane transporters, menaquinone biosynthesis, and complexes I–IV of the eubacterial respiratory chain that functions in the haloarchaeal membrane consisting of diphytanyl isoprene ether lipids. LGT on a massive scale transformed a strictly anaerobic, chemolithoautotrophic methanogen into the heterotrophic, oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarchaeal common ancestor.Halophilic archaebacteria (Haloarchaea) require concentrated salt solutions for survival and can inhabit saturated brine environments such as salt lakes, the Dead Sea, and salterns (1). In rRNA and phylogenomic analyses of informational genes, Haloarchaea always branch well within the methanogens (2–4). Haloarchaea can thus be seen as deriving from methanogen ancestors, but the physiology of methanogens and halophiles could hardly be more different. Methanogens are strict anaerobes, most species are lithoautotrophs that use electrons from H₂ to reduce CO₂ to methane (obligate hydrogenotrophic methanogens), thereby generating a chemiosmotic ion gradient for ATP synthesis in their energy metabolism, although some species can generate methane from reduced C₁ compounds, or acetate in the case of aceticlastic forms (5–7). Their carbon metabolism involves the Wood–Ljungdahl (acetyl-CoA) pathway of CO₂ fixation (5–7). In contrast, Haloarchaea are obligate heterotrophs that typically use O₂ as the terminal acceptor of their electron transport chain, although many can also use alternative electron acceptors such as nitrate in addition to light harnessing via a bacteriorhodopsin-based proton pumping system (8). The evolutionary nature of that radical physiological transformation from anaerobic chemolithoautotroph to aerobic heterotroph is of interest.Many individual reports document that lateral gene transfer (LGT) from eubacteria was involved in the origin of at least some components of haloarchaeal metabolism. These include the operon for gas vesicle formation, which allows Haloarchaea to remain in surface waters (9), the newly identified methylaspartate cycle of acetyl-CoA oxidation (10), various components of the haloarchaeal aerobic respiratory chain (11–18), and proteins involved in the assembly of FeS clusters (19). The sequencing of the first haloarchaeal genome over a decade ago identified some eubacterial genes that possibly could have been acquired by lateral gene transfer (11, 20), and whereas substantial data that would illuminate the origin of haloarchaeal physiology have accumulated since then, those data have not been subjected to comparative evolutionary analysis. Investigating the role of the environment in haloarchaeal genome evolution, Rhodes et al. (21) recently showed that Haloarchaea are indeed far more likely to acquire genes from other halophiles, but they did not address the issues at the focus of our present investigation, namely: How many eubacterial acquisitions are present in haloarchaeal genomes? How was the physiological transformation of methanogens to Haloarchaea affected by LGT? Do those acquisitions trace to the haloarchaeal common ancestor as a single acquisition or not?To discern whether the eubacterial genes in haloarchaeal genomes are the result of multiple independent transfers in individual lineages or the result of a single ancient mass acquisition, here we have analyzed 10 sequenced haloarchaeal genomes—Haloarcula marismortui (22), Halobacterium salinarum (23), Halobacterium sp. (20), Halomicrobium mukohataei (24), Haloquadratum walsbyi (25), Halorhabdus utahensis (26), Halorubrum lacusprofundi (27), Natrialba magadii (28), Natronomonas pharaonis (29), and Haloterrigena turkmmenica (30)—in the context of 65 other archaebacterial and >1,000 eubacterial reference genomes.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.