Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal

Seed priming and sulfur effects on soybean cell membrane stability and yield in saline soil

The objective of this work was to determine the effects of seed priming and sulfur application on cell membrane characteristics, seedling emergence, chlorophyll content and grain yield of soybean (Glycine max) in saline soil. A complete-block design in 4x3 factorial arrangement with three replicates was used to test four types of seed priming (water, auxin, gibberellin and non-priming) and three levels of sulfate availability (0, 70 and 140 kg ha-1 K2SO4). The soil had a silty loam texture with an electrical conductivity of 3.61 ds m-1, a pH of 8.2 and a saturation percentage of about 46%. Seed priming had significant effects on mean emergence rate (MER), emergence percentage, relative water content (RWC) of leaves, relative chlorophyll content, time of maturity, shoot length and grain yield. The highest values for these variables were observed in the priming treatments, except for the time of maturity. Sulfur application had significant effects on MER, shoot length, RWC, membrane injury index and grain yield. Priming treatments provide greater emergence rates and grain yields and interact sinergicaly with sulfur rates.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

Evolution and functional characterisation of the IR chemosensory receptors in the Drosophila larval gustatory system

Chemosensory receptor gene families encode divergent proteins capable of detecting a huge diversity of environmental stimuli that are constantly changing over evolutionary time as organisms adapt to distinct ecological niches. While olfaction is dedicated to the detection of volatile compounds, taste is key to assess food quality for nutritional value and presence of toxic substances. The sense of taste also provides initial signals to mediate endocrine regulation of appetite and food metabolism and plays a role in kin recognition. The fruit fly Drosophila melanogaster is a very good model for studying smell and taste because these senses are very important in insects and because a broad variety of genetic tools are available in Drosophila. Recently, a family of 66 chemosensory receptors, the Ionotropic Receptors (IRs) was described in fruit flies. IRs are distantly related to ionotropic glutamate receptors (iGluRs), but their evolutionary origin from these synaptic receptors is unclear. While 16 IRs are expressed in the olfactory system, nothing is known about the other members of this repertoire. In this thesis, I describe bioinformatic, expression and functional analyses of the IRs aimed at understanding how these receptors have evolved, and at characterising the role of the non-olfactory IRs. I show that these have emerged at the basis of the protostome lineage and probably have acquired their sensory function very early. Moreover, although several IRs are conserved across insects, there are rapid and dramatic changes in the size and divergence of IR repertoires across species. I then performed a comprehensive analysis of IR expression in the larva of Drosophila melanogaster, which is a good model to study taste and feeding mechanisms as it spends most of its time eating or foraging. I found that most of the divergent members of the IR repertoire are expressed in both peripheral and internal gustatory neurons, suggesting that these are involved in taste perception. Finally, through the establishment of a new neurophysiological assay in larvae, I identified for the first time subsets of IR neurons that preferentially detect sugars and amino acids, indicating that IRs might be involved in sensing these compounds. Together, my results indicate that IRs are an evolutionarily dynamic and functionally versatile family of receptors. In contrast to the olfactory IRs that are well-conserved, gustatory IRs are rapidly evolving species-specific receptors that are likely to be involved in detecting a wide variety of tastants. - La plupart des animaux possèdent de grandes familles de récepteurs chimiosensoriels dont la fonction est de détecter l'immense diversité de composés chimiques présents dans l'environnement. Ces récepteurs évoluent en même temps que les organismes s'adaptent à leur écosystème. Il existe deux manières de percevoir ces signaux chimiques : l'olfaction et le goût. Alors que le système olfactif perçoit les composés volatiles, le sens du goût permet d'évaluer, par contact, la qualité de la nourriture, de détecter des substances toxiques et de réguler l'appétit et le métabolisme. L'un des organismes modèles les plus pertinents pour étudier le sens du goût est le stade larvaire de la mouche du vinaigre Drosophila melanogaster. En effet, la principale fonction du stade larvaire est de trouver de la nourriture et de manger. De plus, il est possible d'utiliser tous les outils génétiques développés chez la drosophile. Récemment, une nouvelle famille de 66 récepteurs chimiosensoriels appelés Récepteurs Ionotropiques (IRs) a été découverte chez la drosophile. Bien que leur orogine soit peu claire, ces récepteurs sont similaires aux récepteurs ionotropiques glutamatergiques impliqués dans la transmission synaptique. 16 IRs sont exprimés dans le système olfactif de la mouche adulte, mais pour l'instant on ne connaît rien des autres membres de cette famille. Durant ma thèse, j'ai effectué des recherches sur l'évolution de ces récepteurs ainsi que sur l'expression et la fonction des IRs non olfactifs. Je démontre que les IRs sont apparus chez l'ancêtre commun des protostomiens et ont probablement acquis leur fonction sensorielle très rapidement. De plus, bien qu'un certain nombre d'IRs olfactifs soient conservés chez les insectes, d'importantes variations dans la taille et la divergence des répertoires d'IRs entre les espèces ont été constatées. J'ai également découvert qu'un grand nombre d'IRs non olfactifs sont exprimés dans différents organes gustatifs, ce qui leur confère probablement une fonction dans la perception des goûts. Finalement, pour la première fois, des neurones exprimant des IRs ont été identifiés pour leur fonction dans la perception de sucres et d'acides aminés chez la larve. Mes résultats présentent les IRs comme une famille très dynamique, aux fonctions très variées, qui joue un rôle tant dans l'odorat que dans le goût, et dont la fonction est restée importante tout au long de l'évolution. De plus, l'identification de neurones spécialisés dans la perception de certains composés permettra l'étude des circuits neuronaux impliqués dans le traitement de ces informations.

Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

GLUT8 subcellular localization and absence of translocation to the plasma membrane in PC12 cells and hippocampal neurons.

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Dissection of hormone-responsive plasma membrane to nucleus signaling of Bravis Radix : its role in vascular differentiation and root meristem growth

AbstractPlants continuously grow during their complete life span and understanding the mechanisms that qualitatively regulate their traits remains a challenging topic in biology. The hormone auxin has been identified as a crucial molecule for shaping plant growth, as it has a role in most developmental processes. In the root, the directional, so-called polar transport of auxin generates a peak of concentration that specifies and maintains the stem cell niche and a subsequent gradient of decreasing concentration that also regulates cell proliferation and differentiation. For these reasons, auxin is considered the main morphogen of the root, as it is fundamental for its organization and maintenance. Recently, in Arabidopsis thaliana, a natural variation screen allowed the discovery of BREVIS RADIX (BRX) gene as a limiting factor for auxin responsive gene expression and thus for root growth.In this study, we discovered that BRX is a direct target of auxin that positively feeds back on auxin signaling, as a transcriptional co-regulator, through interaction with the Auxin Response Factor (ARF) MONOPTEROS (MP), modulating the auxin gene response magnitude during the transition between division and differentiation in the root meristem. Moreover, we provide evidence that BRX is activated at the plasma membrane level as an associated protein before moving into the nucleus to modulate cellular growth.To investigate the discrepancy between the auxin concentration and the expression pattern of its downstream targets, we combined experimental and computational approaches. Expression profiles deviating from the auxin gradient could only be modeled after intersection of auxin activity with the observed differential endocytosis pattern and with positive auto- regulatory feedback through plasma- membrane-to-nucleus transfer of BRX. Because BRX is required for expression of certain auxin response factor targets, our data suggest a cell-type-specific endocytosis-dependent input into transcriptional auxin perception. This input sustains expression of a subset of auxin-responsive genes across the root meristem's division and transition zones and is essential for meristem growth. Thus, the endocytosis pattern provides specific positional information to modulate auxin response. RésuméLes plantes croissent continuellement tout au long de leur cycle de vie. Comprendre et expliquer les mécanismes impliqués dans ce phénomène reste à l'heure actuelle, un défi. L'hormone auxine a été identifiée comme une molécule essentielle à la régulation de la croissance des plantes, car impliquée dans la plupart des processus développementaux. Dans la racine, le transport polaire de l'auxine, par la génération d'un pic de concentration, spécifie et maintient la niche de cellules souches, et par la génération d'un gradient de concentration, contrôle la prolifération et la différentiation cellulaire. Puisque l'auxine est essentielle pour l'organisation et la maintenance du système racinaire, il est considéré comme son principal morphogène. Récemment, dans la plante modèle, Arabidopsis thalinana, un criblage des variations génétique a permis d'identifier le gène Brevis radix (BRX) comme facteur limitant l'expression des gènes de réponse à l'auxine et par là même, la croissance de la racine.Dans ce travail, nous avons découvert que BRX est une cible direct de l'auxine qui rétroactive positivement le signalement de l'hormone, agissant ainsi comme un régulateur transcriptionnel à travers l'interaction avec la protéine Monopteros (MP) de la famille des facteurs de réponse à l'auxine (Auxin Responsive Factor, ARF), et modulant ainsi la magnitude de la réponse des gènes reliés à l'auxine durant la division et la différentiation cellulaire dans le méristème de la racine. De plus, nous fournissons des preuves que BRX est activées au niveau de la membrane plasmique, tel une protéine associée se déplaçant à l'intérieur du noyau et modulant la croissance cellulaire.Pour mener à bien l'investigation des divergences entre la concentration de l'auxine et les schémas d'expression de ses propres gènes cibles, nous avons combiné les approches expérimentales et computationnelles. Les profiles d'expressions déviant du gradient d'auxine pourraient seulement être modéliser après intersection de l'activité de l'auxine avec les schémas différentiels d'endocytose observés et les boucles de rétroaction positives et autorégulatrices par le transfert de BRX de la membrane plasmique au noyau. Puisque BRX est requis pour l'expression de certains gènes cibles des facteurs de réponse à l'auxine, nos données suggèrent une contribution dépendante d'une endocytose spécifique au type de cellule dans la perception transcriptionnelle à l'auxine Cette contribution soutient l'expression d'un sous-set de gène de réponse à l'auxine dans la division du méristème racinaire et la zone de transition, et par conséquent, est essentielle pour la croissance méristematique. Ainsi, le schéma d'endocytose fournit des informations positionnelles spécifiques à la modulation de la réponse à l'auxine.

Membrane insertion and antibody recognition of a glycosylphosphatidylinositol-anchored protein: an optical study.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.

Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia.

Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.

Isolation, culture and characterisation of human endothelial progenitor cells

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