Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control

Biological ammonium transporters from the Amt/Mep/Rh superfamily : mechanism, energetics, and technical limitations

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life and is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. Remarkably, despite a high structural conservation in all domains of life, these proteins have gained various biological functions during evolution. It is tempting to hypothesise that the physiological functions gained by these proteins may be explained at least in part by differences in the energetics of their translocation mechanisms. Therefore, in this review, we will explore our current knowledge of energetics of the Amt/Mep/Rh family, discuss variations in observations between different organisms, and highlight some technical drawbacks which have hampered effects at mechanistic characterisation. Through the review we aim to provide a comprehensive overview of current understanding of the mechanism of transport of this unique and extraordinary Amt/Mep/Rh superfamily of ammonium transporters

Biological ammonium transporters : evolution and diversification

Although ammonium is the preferred nitrogen source for microbes and plants, in animal cells it is a toxic product of nitrogen metabolism that needs to be excreted. Thus, ammonium movement across biological membranes, whether for uptake or excretion, is a fundamental and ubiquitous biological process catalysed by the superfamily of the Amt/Mep/Rh transporters. A remarkable feature of the Amt/Mep/Rh family is that they are ubiquitous and, despite sharing low amino acid sequence identity, are highly structurally conserved. Despite sharing a common structure, these proteins have become involved in a diverse range of physiological process spanning all domains of life, with reports describing their involvement in diverse biological processes being published regularly. In this context, we exhaustively present their range of biological roles across the domains of life and after explore current hypotheses concerning their evolution to help to understand how and why the conserved structure fulfils diverse physiological functions

Coexistence of Ammonium Transporter and Channel Mechanisms in Amt-Mep-Rh Twin-His Variants Impairs the Filamentation Signaling Capacity of Fungal Mep2 Transceptors

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin- His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors

Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation

Characterization of organic molecules within solar system analogues

La matière organique est une matière dont les molécules contiennent au moins une liaison chimique carbone-hydrogène. Sur Terre, elle est intrinsèquement liée à la biosphère. Cependant, cette matière n’est pas exclusivement liée aux systèmes vivants. Elle peut être liée à des processus biologiques (photosynthèse), géologiques (pétroles) et abiotiques (aérosols secondaires). Elle présente une diversité dans tout le système solaire : les observations suggèrent la présence d’une chimie organique complexe abiotique dans les nuages de Titan, les océans d’Europe et d'Encelade, les roches des météorites ou encore les glaces cométaires. Les comètes sont les objets qui ont été le moins altérés depuis 4,5 milliards d’années. L’étude de leur composition permet de mieux comprendre les processus à origine de la matière organique sur Terre.- Durant cette thèse, la composition possible d’une comète telle qu’elle pourrait être identifiée lors d’une mission spatiale est étudiée. On suppose que la matière organique d’une comète se répartit en deux phases principales : solide ou gazeuse. L’objectif de cette thèse est d’identifier les corrélations existantes entre la composition en matière organique de ces phases. Dans le premier chapitre on détaille les connaissances actuelles concernant la matière organique et les comètes ainsi que les résultats déjà existants dans ce domaine. Dans le deuxième, on décrit les protocoles mis au point durant la thèse pour l’analyse de la phase solide. Dans le troisième, le protocole expérimental permettant l’analyse de la phase gazeuse est présenté. Enfin, dans le dernier, les premiers résultats et premières corrélations mises en évidence sont exposésOrganic matter is at least composed of one carbon-hydrogen bond. It is highly linked to biological activity on earth. However, we know from two centuries old studies that this matter is not exclusively produced from living systems. Three main production and alteration processes can be identified: biological (photosynthesis…), geological (petroleum…) and abiotic (secondary aerosols…). Titan's clouds, Europa's and Enceladus' oceans, meteoritic rocks or even cometary ices… All over the solar system a surprisingly complex abiotic organic chemistry has been found. Among all of these wonderful places, comets are the ones that remained unchanged for almost 4.5 billion years. Studying their composition provides a better understanding of both the solar system formation and the origin of organic matter on Earth. During this PhD thesis, the expected comet composition as space missions would study them is studied. Thus, an original experimental protocol allowing the recovery and identification of organic matter from synthetic comets is developed. The keystone of this study is that organic matter of a comet is divided into two main phases: solid or gaseous. The main objective is to identify the existing correlations between both of them. This manuscript is organized into four chapters. First, the current knowledge linking organic matter and comets as well as the first results in this field is detailed. Second, the developed protocols for cometary analogs solid phase analysis as well as developed tools is described. Third, the gas phase recovery experimental protocol is presented. Lastly, the first results and the first highlighted correlations are show

Identify low mass volatile organic compounds from cometary ice analogs using gas chromatography coupled to an Orbitrap mass spectrometer associated to electron and chemical ionizations

International audienc

In vivo functional characterization of the Escherichia coli ammonium channel AmtB: Evidence for metabolic coupling of AmtB to glutamine synthetase

F.I. 4.3450 C.I. 53info:eu-repo/semantics/publishe