Glucose Transport in the Extremely Thermoacidophilic Sulfolobus solfataricus Involves a High-Affinity Membrane-Integrated Binding Protein

The archaeon Sulfolobus solfataricus grows optimally at 80°C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0.43 µM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose

The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium

AbstractRhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems

Phosphoenolpyruvate-Dependent Fructose Phosphotransferase System of Rhodopseudomonas sphaeroides: Purification and Physicochemical and Immunochemical Characterization of a Membrane-Associated Enzyme I

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called “soluble factor” (SF). SF has been partially purified by a combination of hydrophobic interaction and ion-exchange and gel-permeation chromatography. SF is similar to Escherichia coli enzyme I in its molecular characteristics and enzymatic properties. It has a molecular weight of 85 000 and readily dimerizes. Phosphoenolpyruvate and Mg2+ stabilize the dimer. The enzyme catalyzes the conversion of phosphoenolpyruvate into pyruvate and becomes phosphorylated in the process. The phosphoryl group is subsequently transferred to fructose in the presence of R. sphaeroides membranes. SF substitutes for E. coli enzyme I in fructose or glucose phosphorylation with E. coli enzyme II and HPr. The activities of SF with the R. sphaeroides PTS and the E. coli PTS reside on structurally distinct molecules as shown by their response to limited proteolytic digestion and by immunochemical studies. The activity of SF with the E. coli PTS arises during the isolation procedure and is most likely due to the removal of HPr-like protein from SF.

Slow fusion of liposomes composed of membrane-spanning lipids

The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated. These lipids span the entire membrane and form single monolayer liposomes in aqueous media. In the presence of calcium-phosphate, slow mixing of the aqueous liposome contents and membrane lipids occurred, demonstrating that these liposomes are fusion-competent. The fusion process was essentially nonleaky. The rate of fusion increased with the pH and the concentration of calcium and phosphate. Fusion resulted in an increase of the size of the liposomes. These data demonstrate that a monolayer organization of lipids in a membrane does not per se interfere with membrane fusion competence.

Stability and proton-permeability of liposomes composed of archaeal tetraether lipids

Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower proton permeability than liposomes composed of diester lipids originating from the mesophilic bacterium Escherichia coli or the thermophilic bacterium Bacillus stearothermophilus. With increasing temperature, the stability decreased and the proton permeability increased for all liposomes. Liposomes composed from tetraether lipids, however, remain the most stable. These data suggest these liposomes retain the rigidity of the cytoplasmic membrane of S. acidocaldarius needed to endure extreme environmental growth conditions.</p

In vitro pore-forming activity of the lantibiotic nisin. Role of protonmotive force and lipid composition

Nisin is a lantibiotic produced by some strains of Lactococcus luctis subsp. lactis. The target for nisin action is the cytoplasmic membrane of Gram-positive bacteria. Nisin dissipates the membrane potential (Δψ) and induces efflux of low-molecular-mass compounds. Evidence has been presented that a Δψ is needed for nisin action. The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein. Nisin-induced efflux of carboxyfluorescein was observed in the absence of a Δψ from liposomes composed of Escherichia coli lipids or dioleoylglycerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios. The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios. Both Δψ (inside negative) and ΔpH (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values. Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory. It is concluded that a Δψ is not essential, but that the total protonmotive force stimulates the action of nisin.