Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1

The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.

Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1

The aerobactin-mediated iron uptake system encoded by pColV-K30 and other ColV plasmids has been associated with the ability of Escherichia coli strains to cause disease. We investigated whether the pColV-K30 aerobactin system is present in E. coli K1 VW187 isolated from a human neonate with meningitis. This strain exhibited a functional aerobactin-mediated iron uptake system, as assessed by a cross-feeding bioassay and by its sensitivity to cloacin, a bacteriocin that recognizes the outer membrane receptor for iron-aerobactin complexes. By using a variety of techniques, we could not find any plasmid harboring the aerobactin genes. Hybridization of restriction endonuclease-cleaved chromosomal DNA from strain VW187 with various clones containing subsets of the pColV-K30 aerobactin region showed that the aerobactin genes were located on a 10.5-kilobase-pair chromosomal HindIII restriction fragment which also contained IS1-like insertion sequences. The chromosomal aerobactin region showed a high degree of conservation when compared with the homologous region in plasmid pColV-K30, although it was located on a different restriction endonuclease site environment.

High-affinity NO(3-)-H+ cotransport in the fungus Neurospora:induction and control by pH and membrane voltage

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO3(-)-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 microM NO3-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO3-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO3- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 microM NO3-; and it was suppressed when NH4+ was present during the first, inductive exposure to NO3-. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (-400 to +100 mV). Measurements of NO3- uptake using NO3(-)-selective macroelectrodes indicated a charge stoichiometry for NO3- transport of 1(+):1(NO3-) with common K(m) and Jmax values around 25 microM and 75 pmol NO3- cm-2sec-1, respectively, and combined measurements of pHo and [NO3-]o showed a net uptake of approx. 1 H+ with each NO3- anion. Analysis of the NO3- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pHo and [NO3-]o. Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pHo of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO3-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H+]o. These, and additional results are consistent with a charge-coupling stoichiometry of 2(H+) per NO3- anion transported across the membrane, and implicate a carrier cycle in which NO3- binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO3- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO3- transport; finally, they distinguish metabolite repression of NO3- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

Does unidirectional vesicular transport occur in retinal vessels?

This paper challenges the hypothesis that the smooth 80 nm plasmalemmal caveolae found in abundance at the abluminal aspect of the endothelium in retinal blood vessels participate in a unidirectional vesicular transport mechanism. Evidence is presented which indicates that horseradish peroxidase, when introduced to the extracellular space of the retina via the vitreous body, may enter the intravascular compartment through junctional incompetence which occurs at or after enucleation of the eye. It is proposed that the plasmalemmal caveolae at the abluminal plasma membrane of endothelial cells in retinal blood vessels are static structures which facilitate the transport of small solutes and ions across the blood retinal barrier.

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.

Burkholderia cenocepacia and Salmonella enterica ArnT proteins that transfer 4-amino-4-deoxy-L-arabinose to lipopolysaccharide share membrane topology and functional amino acids

We recently demonstrated that incorporation of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of lipopolysaccharide (LPS) is required for transport of LPS to the outer membrane and viability of the Gram-negative bacterium Burkholderia cenocepacia. ArnT is a membrane protein catalyzing the transfer of l-Ara4N to the LPS molecule at the periplasmic face of the inner membrane, but its topology and mechanism of action are not well characterized. Here, we elucidate the topology of ArnT and identify key amino acids that likely contribute to its enzymatic function. PEGylation assays using a cysteineless version of ArnT support a model of 13 transmembrane helices and a large C-terminal region exposed to the periplasm. The same topological configuration is proposed for the Salmonella enterica serovar Typhimurium ArnT. Four highly conserved periplasmic residues in B. cenocepacia ArnT, tyrosine-43, lysine-69, arginine-254 and glutamic acid-493, were required for activity. Tyrosine-43 and lysine-69 span two highly conserved motifs, 42RYA44 and 66YFEKP70, that are found in ArnT homologues from other species. The same residues in S. enterica ArnT are also needed for function. We propose these aromatic and charged amino acids participate in either undecaprenyl phosphate-l-Ara4N substrate recognition or transfer of l-Ara4N to the LPS.

Arsenite transport into paddy rice (Oryza sativa) roots

Here the mechanism of arsenite transport into paddy rice (Oryza sativa) roots, uptake of which is described by Michaelis-Menten kinetics, is reported. A recent study on yeast (Saccharomyces cerevisiae) showed that undissociated arsenite (its pK_a is 9.2) was transported across the plasma membrane via a glycerol transporting channel. To investigate whether the same mechanism of transport was involved for rice, competitive studies with glycerol, which is transported into cells via aquaporins, were performed. Glycerol competed with arsenite for transport in a dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were the same. Arsenate transport was unaffected by glycerol, confirming that arsenate and arsenite are taken up into cells by different mechanisms. Antimonite, an arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also competed with arsenite transport in a dose-dependent manner, providing further evidence that arsenite is transported into rice roots via glycerol transporting channels. Mercury (Hg²⁺) inhibited both arsenite and arsenate uptake, suggesting that inhibition of influx was due to general cellular stress rather than the specific action of Hg²⁺ on aquaporins. Arsenite uptake by pea (Pisum sativum) and wheat (Triticum aestivum) was also described by Michaelis-Menten kinetics.

High-affinity NO3 --H+ cotransport in the fungus Neurospora:Induction and control by pH and membrane voltate

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO₃ ^- challenge and to quantify transport activity. The NO₃ ^--associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO₃ ^--free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μM NO₃ ^-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity mensurable approx. 100 min after first exposure to NO₃ ^-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO₃ ^- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 μM NO₃ ^-; and it was suppressed when NH₄ ⁺, was present during the first, inductive exposure to NO₃ ^-. Voltage clamp measurements carried out immediately before and following NO₃ ^- additions showed that the NO₃ ^--evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages -400 to +100 mV). Measurements of NO₃ ^- uptake using NO₃ ^--selective macroelectrodes indicated a charge stoichiometry for NO₃ ^- transport of 1(+):1(NO₃ ^-) with common K(m) and J(max) values around 25 μM and 75 pmol NO₃ ^- cm^-2sec^-1, respectively, and combined measurements of pH(o) and [NO₃ ^-](o) showed a net uptake of approx. 1 H⁺ with each NO₃ ^- anion. Analysis of the NO₃ ^- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pH(o) and [NO₃ ^-](o). Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH(o) of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO₃ ^-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H⁺](o). These, and additional results are consistent with a charge-coupling stoichiometry of 2(H⁺) per NO anion transported across the membrane, and implicate a carrier cycle in which NO binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO₃ ^- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO₃ ^- transport; finally, they distinguish metabolite repression of NO₃ ^- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Modelling and optimization of the ion exchange membrane bioreactor for removal of anionic pollutants from drinking water streams

Dissertação para obtenção do Grau de Doutor em Engenharia Química, especialidade de Engenharia Bioquímica

Transport of methotrexate by the in vitro isolated rabbit proximal tubule.

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.