Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 μM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes

Do All Lives Have the Same Value? Support for International Military Interventions as a Function of Political System and Public Opinion of the Target States

This research examined the support for international military interventions as a function of the political system and the public opinion of the target country. In two experiments, we informed participants about a possible military intervention by the international community towards a sovereign country whose government planned to use military force against a secessionist region. They were then asked whether they would support this intervention whilst being reminded that it would cause civilian deaths. The democratic or nondemocratic political system of the target country was experimentally manipulated, and the population support for its belligerent government policy was either assessed (Experiment 1) or manipulated (Experiment 2). Results showed greater support for the intervention when the target country was nondemocratic, as compared to the democratic and the control conditions, but only when its population supported the belligerent government policy. Support for the external intervention was low when the target country was democratic, irrespective of national public opinion. These findings provide support for the democracy-as-value hypothesis applied to international military interventions, and suggest that civilian deaths (collateral damage) are more acceptable when nondemocratic populations support their government's belligerent policy

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented