Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida , as well as Vibrio cholerae . Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β-mercaptoethanol and by a dsbA mutant of V. cholerae , indicating that disulphide bond formation may not be essential for release.     

Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.     

Determination of low boron concentrations in nutrient solution

To study boron (B) deficiency and toxicity in plants in flowing solution culture, it is necessary to establish a wide range of solution B concentrations. The ability of inductively coupled plasma atomic emisson spectrophotometry (ICPAES) to determine solution B concentrations ranging from 0.15 M to 925 M was investigated. The reliablity of B concentration determination in nutrient solutions containing <10 M B is poor. A technique, involving sorption by a B-specific resin (Amberlite-743), was established to concentrate the B present in low B nutrient solutions and to enable reliable measurement using ICPAES following elution from the resin. Acceptable, reproducible recoveries of B from low B solutions, containing known concentrations of B, were obtained using this technique. The technique enables the imposition, monitoring and maintenance of solution B concentrations well below the direct detection limits of ICPAES and colorimetric procedures. ei]L V Kochian     

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein

The plasma membrane of rat erythrocytes contains a 47-kDa glycoprotein that binds the channel-forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N-terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N-termini of a group of membrane proteins that are thought to be involved in ADP-ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C-terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol-specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells' sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin-binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.     

Amyloid-beta protein oligomer at low nanomolar concentrations activates microglia and induces microglial neurotoxicity

Neuroinflammation and associated neuronal dysfunction mediated by activated microglia play an important role in the pathogenesis of Alzheimer disease (AD). Microglia are activated by aggregated forms of amyloid-β protein (Aβ), usually demonstrated in vitro by stimulating microglia with micromolar concentrations of fibrillar Aβ, a major component of amyloid plaques in AD brains. Here we report that amyloid-β oligomer (AβO), at 5-50 nm, induces a unique pattern of microglia activation that requires the activity of the scavenger receptor A and the Ca(2+)-activated potassium channel KCa3.1. AβO treatment induced an activated morphological and biochemical profile of microglia, including activation of p38 MAPK and nuclear factor κB. Interestingly, although increasing nitric oxide (NO) production, AβO did not increase several proinflammatory mediators commonly induced by lipopolyliposaccharides or fibrillar Aβ, suggesting that AβO stimulates both common and divergent pathways of microglia activation. AβO at low nanomolar concentrations, although not neurotoxic, induced indirect, microglia-mediated damage to neurons in dissociated cultures and in organotypic hippocampal slices. The indirect neurotoxicity was prevented by (i) doxycycline, an inhibitor of microglia activation; (ii) TRAM-34, a selective KCa3.1 blocker; and (iii) two inhibitors of inducible NO synthase, indicating that KCa3.1 activity and excessive NO release are required for AβO-induced microglial neurotoxicity. Our results suggest that AβO, generally considered a neurotoxin, may more potently cause neuronal damage indirectly by activating microglia in AD.     

Isolated EF-loop III of calmodulin in a scaffold protein remains unpaired in solution using pulsed-field-gradient NMR spectroscopy

Calmodulin (CaM) is a trigger calcium-dependent protein that regulates many biological processes. We have successfully engineered a series of model proteins, each containing a single EF-hand loop but with increasing numbers of Gly residues linking the EF-hand loop to a scaffold protein, cluster of differentiation 2 (CD2), to obtain the site-specific calcium-binding ability of a protein with EF-hand motifs without the interference of cooperativity. Loop III of calmodulin with two Gly linkers in CD2 (CaM-CD2-III-5G) has metal affinities with K(d) values of 1.86 x 10(-4) and 5.8 x 10(-5) M for calcium and lanthanum, respectively. The oligomeric states of the CD2 variants were examined by pulsed-field-gradient nuclear magnetic resonance (PFG NMR). The diffusion coefficient values of CD2 variants are about 11.1 x 10(-7) cm(2)/s both in the presence and absence of metal ions, which are the same as that of wild-type CD2. This suggests that the isolated EF-loop III of calmodulin inserted in the scaffold protein is able to bind calcium and lanthanum as a monomer, which is in contrast to the previous observation of the EF-hand motif. Our results imply that additional factors that reside outside of the EF-loop III may contribute to the pairing of EF-hand motifs of calmodulin. This result is of interest as it opens up the way for studying the ion-binding properties of isolated EF-hands, which in turn can answer important questions about the properties of EF-hands, the large and important group of calcium-binding signaling proteins.     

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

The periplasmic maltose-binding protein modifies the channel-forming characteristics of maltoporin.

Maltoporin, a protein spanning Escherichia coli outer membranes, modifies electrical conductance of membranes due to its channel-forming properties. This observation was made by conductance measurements across planar bilayers which were derived from unextracted, isolated outer membrane vesicles using a porin-deficient E. coli strain. Alternatively, proteoliposomes reconstituted with detergent-solubilized homogeneous maltoporin and phospholipids were used. With either membrane preparation, channel conductance was observed, although no discrete conductance levels were detected. The presence of lipopolysaccharide, a bacterial glycolipid, was not required, nor did it affect channel activity. In the presence of the water-soluble periplasmic maltose-binding protein, conductance fluctuations occurred in discrete steps, demonstrating opening and closing events of channels. Multiple step sizes (1/3, 2/3 and 1 ns in 1 M KCl) in single channel traces suggest cooperative opening and closing of up to three channels. The action of maltose-binding protein is highly asymmetrical, and its affinity to maltoporin is very high (KD = 1.5 X 10(-7) M). Association of maltose-binding protein to maltoporin shifts, for a given polarity, the equilibrium between open and closed states in favour of closed states. This result matches earlier in vivo studies, and supports the physiological significance of the observations made.     

Diffusion of DNA at very low concentrations

The diffusion behavior of DNA samples of molecular weights between 1 × 10⁶ and 25 × 10⁶ Daltons was investigated under standard conditions at mean concentrations c? between 0.0009 and 0.017 g/dl. Special techniques described previously were used and supplemented. The sensitivity required was accomplished by multiple passage through the sample cells (effective path length of 10–45 cm) and application of the Gouy interference method. The maximum DNA refraction index difference has been determined more precisely from Gouy interference fringes by applying a systematic variation procedure and a linear-plot criterion. Convection was prevented by a temperature constancy better than 0.002°C/day, vibrationless operation, and by application of a slight density gradient of heavy water, which also improved the boundary-forming procedure. The corresponding optical HDO gradient was compensated. The concentration dependence of the DNA diffusion coefficient average D_A was found to be positive and very small at extremely low concentrations, that is, below c? = 0.008 g/dl, for the sample of highest molecular weight investigated. With beginning penetration of different DNA molecules, D_A increases markedly. The diffusion constant averages of our polydisperse samples will be corrected for monodisperse subfractions in a following paper. The resulting molecular weights M from diffusion and sedimentation constants (D⁰, s⁰) together with data from literature are the basis of new s⁰–M, D⁰ ? M, and [η]–M relations for monodisperse DNA samples.     

Aquatic nitrogen transformations at low oxygen concentrations

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Free human mitochondrial GrpE is a symmetric dimer in solution

The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.     

Aerobic microbial growth at low oxygen concentrations

Sterilizable membrane probes were used to study the relation between oxygen concentration and respiration rate in Candida utilis growing on acetate. When the organism was grown in a continuous fermentor at various dissolved oxygen concentrations (0.23 x 10(-6) to 32 x 10(-6)m), with time allowed for full adaptation to each oxygen concentration, the relationship between oxygen concentration and growth rate simulated Michaelis-Menten behavior, giving an apparent K(m) for oxygen of 1.3 x 10(-6)m. When respiration rate was measured at various oxygen concentrations without allowing time for adaptation, it was found that the respiration rate was directly proportional to O(2) concentration at low O(2) concentrations, and independent of O(2) concentration at high O(2) concentrations. Transition from one type of behavior to the other was fairly abrupt. The respiration rate in the presence of excess oxygen depended on the O(2) concentration at which the cells were grown, but the rate at low O(2) concentrations did not. There was evidence that, at low oxygen concentrations, oxygen diffusion through the cell substance limits respiration rate, at least in part.     

Nutritional versatility of a starch-utilizing Flavobacterium at low substrate concentrations.

A starch-utilizing yellow-pigmented bacterium, isolated from tap water, was tested for the utilization of 64 natural compounds at a concentration of 1 g/liter by measuring colony growth on agar media. Only 12 carbohydrates and glycerol promoted growth. Growth experiments with the organism in pasteurized tap water supplied with mixtures of substrates at concentrations of 1 or 10 micrograms of C of each substrate per liter, followed by separate experiments with a number of carbohydrates at 10 micrograms of C per liter showed that of these 64 natural compounds only sucrose, maltose, raffinose, starch, and glycerol promoted growth at very low concentrations. Also maltotriose, -tetraose, -pentaose, -hexaose, and stachyose, which were not included in the mixtures, enhanced growth, and generation times of 3 to 5 h at 10 micrograms of C per liter were observed. The organism, which was tentatively identified as a Flavobacterium species, thus appeared to be highly specialized in the utilization of glycerol and a number of oligo- and polysaccharides at very low concentrations.     

Considerations in the determination of boron at low concentrations

Experimental evidence now supports the nutritional essentiality of boron (B) in some biological systems, and accordingly, the need for reliable analytical B data is increasing. However, the accurate determination of B in biological materials is a formidable challenge at low concentrations (<1 mg B/kg). Recent studies still show significant analytical discrepancies in the analysis of animal tissues and fluids, despite the development of instrumental techniques such as TIMS, ICP-MS, ICP-ES, ICAP, SIMS, NA-MS, PGAA, NRA, and so forth, which have demonstrated detection limits approaching or exceeding (μgB/kg concentrations. Since boric acid is both volatile and ubiquitous in nature, the chemical and physical pathways for B contamination and its loss are manifold, especially during sample preparation. An added obstacle is the inadequacy of biological reference materials certified for B below mg B/kg. With an emphasis toward sample preparation and ICP-MS analysis, examples are provided in this article to help the analyst avoid common problems associated with the analysis of B from biological sources. Topics that are discussed include contamination from Teflon vessels during microwave digestion, losses owing to freeze-drying, B isotopic variations, standards preparation, reagent backgrounds, and instrumental interferences.     

Capture of arginine at low concentrations by a marine psychrophilic bacterium

The cells of the marine bacterium Ant-300 were found to take up arginine when this substrate was at low concentrations. The cells possessed an uptake system(s) that specifically transported l-arginine. The kinetic parameters for uptake appeared to differ when the cells were exposed to nanomolar and micromolar concentrations of the amino acid. Uptake over this concentration range functioned in the absence of an exogenous energy source, even after the cells had been preincubated in unsupplemented artificial seawater. Respiratory activity appeared to be a more important driving force for arginine uptake than adenosine 5'-triphosphate hydrolysis. The cells also exhibited chemotaxis toward l-arginine. The minimum arginine concentration needed to elicit a chemotactic response was between 10 and 10 M. It is proposed that the capture of arginine by cells of Ant-300 in nutrient-depleted waters, which are typical of the open ocean, proceeds via high-affinity active transport, whereas in substrate-enriched seawater, capture involves chemotaxis and an active transport mechanism with reduced affinity for the substrate.     

The kinetics of cellulose dissolution in sodium hydroxide solution at low temperatures

The dissolution kinetics of cellulose in sodium hydroxide in the presence and absence of urea at low temperature was studied. High molecular weight cotton linter with degree of polymerization of 850 was used for dissolution study. The cotton linter was separated from the dissolution slurry at different dissolution times, and the change of the crystal structure of cotton linter was characterized by Powder X-Ray Diffraction. The rate of decrystallization of cellulose was obtained and the activation energy for cellulose decrystallization in sodium hydroxide solution was derived using Eyring equation. The effect of urea additive was discussed.