Anion exchange reactions in bacteria

Bacterial anion exchange now includes both carboxylate-linked reactions, in which there is an antiport of mono- and dicarboxylic acids, and Pi-linked reactions that build on phosphate (Pi) and organic phosphates. To illustrate the general features of this expanding class, this article discussed the biochemistry, physiology, and molecular biology of Pi-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. Kinetic and biochemical analysis suggsts that Pi-linked exchangers have a bifunctional active site that accepts a pair of negative charges. For this reason, exchange stoichiometry moves between the limits of 2:1 and 2:2 to reflect the ratio of mono- and divalent substrates at either membrane surface. This results in a particularly interesting reaction sequencein vivo, where, because cytosolic pH is relatively alkaline, one can expect the asymmetric exchange of two monovalent G6P anions against a single divalent G6P. In this way, an otherwise futile self-exchange of G6P gives a net flux driven (indirectly) by the pH gradient. Despite this biochemical and physiological complexity, Pi-linked carriers resemble all other secondary carriers at a molecular level. Indeed, sequence analysis leads one to infer a common (albeit low resolution) structural theme in which each functional unit has two sets of six trans-membrane helices separated by a central hydrophilic loop. Present examples show that this topology can derive from either a single protein, as is typical in bacteria, or from pairs of identical subunits, as found in mitochondria and chloroplasts. The finding of this common structure should make it possible to build detailed structural models that have implications for all membrane carrier proteins.     

Impaired cell volume regulation in taurine deficient cultured astrocytes

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%–16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30–35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.     

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

Clustering of halophytic species from Cyprus based on ionic contents

This paper presents the work conducted on the chemical constituents of some common and widely distributed halophyte taxa from Cyprus with the aim that these studies will help in the evaluation of halophytes for different economical purposes. The plant species of Crithmum maritimum L., Limbarda crithmoides (L.) Dumort, Atriplex portulacoides L., Salsola kali L., Atriplex halimus L., Limonium oleifolium Mill., L. meyeri (Boiss.) Kuntze; and Tetraena alba (L.f.) Beier & Thulin were collected in the middle of July. The shoot tissue and leaf samples were collected from the natural habitats and left for drying under air circulation followed by placing them in oven at 60 °C for 96 hours. The material was crushed using mortar and pestle and subjected to an analysis of macro- and micro-nutrients and biochemical compounds. K⁺/Na⁺ in the leaf tissues of the dicot species showed relatively high values depicting their behavior as Na+ includes but very low Cl- levels were recorded. Out of the species investigated here in 4 TFAA content was rather high. Values ranging from 0.5% to 1% dry weight were exhibited in one species. However, only 3 species showed very low TFAA values. Later may be due to low nitrogen availability in their environment. The phenetic analyses of eight halophyte species performed on the data matrix using Ntsys-pc program version 2.1 revealed that, cluster analysis of the overall results obtained here leads to 2 clusters. This discrimination appears to be as a result of their different abilities to accumulate either proline or glycine betaine.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Observing the osmophobic effect in action at the single molecule level

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments.     

Systemic response to aphid infestation by <Emphasis Type="Italic">Myzus persicae</Emphasis> in the phloem of <Emphasis Type="Italic">Apium graveolens</Emphasis>

Little is known about the molecular processes involved in the phloem response to aphid feeding. We investigated molecular responses to aphid feeding on celery (Apium graveolenscv. Dulce) plants infested with the aphid Myzus persicae, as a means of identifying changes in phloem function. We used celery as our model species as it is easy to separate the phloem from the surrounding tissues in the petioles of mature leaves of this species. We generated a total of 1187 expressed sequence tags (ESTs), corresponding to 891 non-redundant genes. We analysed these ESTs in silico after cDNA macroarray hybridisation. Aphid feeding led to significant increase in RNA accumulation for 126 different genes. Different patterns of deregulation were observed, including transitory or stable induction 3 or 7days after infestation. The genes affected belonged to various functional categories and were induced systemically in the phloem after infestation. In particular, genes involved in cell wall modification, water transport, vitamin biosynthesis, photosynthesis, carbon assimilation and nitrogen and carbon mobilisation were up-regulated in the phloem. Further analysis of the response in the phloem or xylem suggested that a component of the response was developed more specifically in the phloem. However, this component was different from the stress responses in the phloem driven by pathogen infection. Our results indicate that the phloem is actively involved in multiple adjustments, recruiting metabolic pathways and in structural changes far from aphid feeding sites. However, they also suggest that the phloem displays specific mechanisms that may not be induced in other tissues.EST, macroarray and clustering data are available from our website [http://www-biocel.versailles.inra.fr/phloem]. Data deposition: The sequences reported in this paper have been deposited in the Genbank database (Accession nos.: AY607692-AY607700, AY611007, CN253939-CN255151, CV512445-CV512447 and CV651120-CV651121).