Bacterial ApbC can bind and effectively transfer iron-sulfur clusters

The metabolism of iron-sulfur ([Fe-S]) clusters requires a complex set of machinery that is still being defined. Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. ApbC is a 40.8 kDa homodimeric ATPase and as purified contains little iron and no acid-labile sulfide. An [Fe-S] cluster was reconstituted on ApbC, generating a protein that bound 2 mol of Fe and 2 mol of S (2-) per ApbC monomer and had a UV-visible absorption spectrum similar to known [4Fe-4S] cluster proteins. Holo-ApbC could rapidly and effectively activate Saccharomyces cerevisiae apo-isopropylmalate isolomerase (Leu1) in vitro, a process known to require the transfer of a [4Fe-4S] cluster. Maximum activation was achieved with 2 mol of ApbC per 1 mol of apo-Leu1. This article describes the first biochemical activity of ApbC in the context of [Fe-S] cluster metabolism. The data herein support a model in which ApbC coordinates an [4Fe-4S] cluster across its dimer interface and can transfer this cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function recently deduced for its eukaryotic homologues.     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.     

An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Phylogenetic and Functional Heterogeneity of Sediment Biofilms along Environmental Gradients in a Glacial Stream

We used in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes concurrently with measurements of bacterial carbon production, biomass, and extracellular polymeric substances (EPS) to describe the bacterial community in sediments along a glacial stream. The abundance of sediment-associated Archaea, as detected with the ARCH915 probe, decreased downstream of the glacier snout, and a major storm increased their relative abundance by a factor of 5.5 to 7.9. Bacteria of the Cytophaga-Flavobacterium group were also sixfold to eightfold more abundant in the storm aftermath. Furthermore, elevated numbers of Archaea and members of the Cytophaga-Flavobacterium group characterized the phylogenetic composition of the supraglacial ice community. We postulate that glacial meltwaters constitute a possible source of allochthonous bacteria to the stream biofilms. Although stream water temperature increased dramatically from the glacier snout along the stream (3.5 km), sediment chlorophyll a was the best predictor for bacterial carbon production and specific growth rates along the stream. Concomitant with an increase in sediment chlorophyll a, the EPS carbohydrate-to-bacterial-cell ratio declined 11- to 15-fold along the stream prior to the storm, which is indicative of a larger biofilm matrix in upstream reaches. We assume that a larger biofilm matrix is required to assure prolonged transient storage and enzymatic processing of allochthonous macromolecules, which are likely the major substrate for microbial heterotrophs. Bacteria of the Cytophaga-Flavobacterium cluster, which are well known to degrade complex macromolecules, were most abundant in these stream reaches. Downstream, higher algal biomass continuously supplies heterotrophs with easily available exudates, therefore making a larger matrix unnecessary. As a result, bacterial carbon production and specific growth rates were higher in downstream reaches.     

True Chemotaxis in Oxygen Gradients of the Sulfur-Oxidizing Bacterium Thiovulum majus

Observations of free-swimming Thiovulum majus cells show that these bacteria exhibit a phobic response as well as true chemotaxis in oxygen gradients. Both phenomena of their chemotactic behavior are integrated into a single model of helical klinotaxis, which is demonstrated by computer simulations.     

Robotized time-lapse imaging to assess in-planta uptake of phenylurea herbicides and their microbial degradation

Two key physiological parameters of plant leaves, photosynthesis and transpiration, can be continuously monitored by, respectively, chlorophyll a fluorescence imaging and thermography. These non-contact techniques immediately visualize any local stress or treatment affecting either photosynthetic efficiency or water status. Photosystem II-inhibiting herbicides, including the phenylurea derivatives diuron and linuron, cause a marked increase in chlorophyll a fluorescence several days before appearance of chlorosis. Here, bioprotection through microbial degradation of linuron in the feeding solution of common bean plants ( Phaseolus vulgaris L.) was monitored by the absence of an increase in chlorophyll a fluorescence in primary leaves. The different treatments and repeats were imaged sequentially at 2 h intervals using a robotized system with thermal, fluorescence and video cameras. Chlorophyll fluorescence imaging visualized the effect of linuron transported by the transpiration stream earlier than thermography. In addition, local effects and transport after topical application of diuron were recorded presymptomatically in tobacco ( Nicotiana tabacum L.) and Arabidopsis thaliana (L.) Heynh. Thermal imaging clearly monitored localized stomatal closure, coinciding with the first increase in chlorophyll fluorescence, at the sites of diuron treatment. In conclusion, the robotized chlorophyll a fluorescence set-up permits fully reliable, early high-contrast visualization for bioremediation purposes.     

The genome composition of hexaploid Psammopyrum athericum and octoploid Psammopyrum pungens (Poaceae: Triticeae).

The genomic constitution of two species in the genus Psammopyrum, i.e., Ps. athericum (2n = 6x = 42) and Ps. pungens (2n = 8x = 56), was studied by genomic in situ hybridization (GISH). In Ps. athericum, one diploid chromosome set hybridized to a genomic probe from Pseudoroegneria ferganensis (St genome), one diploid set to a probe from Agropyron cristatum (P genome), and one diploid set to a probe from Thinopyrum junceiforme (EbEe genomes) or Th. bessarabicum (Eb genome). Substituting the St-genome probe with an L-genome probe from Festucopsis serpentinii resulted in exactly the same hybridization pattern, suggesting a genomic constitution of EStP or ELP for Ps. athericum. The same probes used on Ps. pungens showed two diploid sets of chromosomes hybridizing to the St-genome probe, one diploid set hybridizing to the P-genome probe, and one diploid set hybridizing to the EbEe-genome probe. The L-genome probe hybridized to approximately 14 of the chromosomes that were labeled by the St-genome probe. Hence the genomic constitution for Ps. pungens is proposed to be EStStP or EStLP.