The effects of ionophores on tetracycline accumulation in Escherichia coli cells were investigated in the presence of polymyxin B nonapeptide. Accumulation was inhibited by nigericin but not by valinomycin. Tetracycline accumulation was stimulated by decreasing the pH of the medium and inhibited by the addition of magnesium ions. These results indicated that tetracycline enters cells through diffusion as a protonated form (TH2) and is accumulated as a membrane-impermeable magnesium-tetracycline chelate complex (THMg+). This noncarrier diffusion hypothesis was confirmed by the fact that tetracycline accumulated in protein-free liposomes through an artificially imposed pH difference.Tetracycline is a broad-spectrum antibiotic which inhibits protein synthesis (6). Since the target of tetracycline is intracellular ribosomes, tetracycline must cross the cell membrane of bacteria to gain access to the target. The pathway for tetracycline through the outer membrane of gram-negative bacteria comprises porin pores (8). On the other hand, there is no consensus as to the pathway through the cytoplasmic membrane. It is known that tetracycline accumulates in sensitive cells of both gram-positive (12) and gram-negative bacteria (7, 14) in an energy-dependent manner. Smith and Chopra (17) reported that both proton motive force and phosphate-bond energy were involved in tetracycline accumulation in Escherichia coli, whereas McMurry et al. (13) reported that proton motive force alone could energize tetracycline uptake. On the other hand, on the basis of the effects of ionophores, Munske et al. (15) claimed that tetracycline uptake by Streptococcus faecalis is driven by a transmembrane pH gradient (ApH) and not by a transmembrane electrical potential (A4i) or by phosphate-bond energy.These investigations indicated the possible presence of a carrier for tetracycline, but no tetracycline carrier has been identified. In contrast, Argast and Beck (1, 2) showed that tetracycline enters the cytoplasm through simple diffusion because of the nonsaturability of tetracycline uptake and presented evidence for diffusion through phospholipid bilayers. The question is how such simple diffusion leads to the accumulation of tetracycline in cells.In contrast to gram-positive bacteria, it has been difficult to investigate the effects of ionophores on the cytoplasmic membrane of gram-negative bacteria because of the outer membrane barrier. In this work, we succeeded in elucidating the effects of ionophores on tetracycline uptake by intact E. coli cells by using polymyxin B nonapeptide (PMBN), which is an outer membrane-specific permeabilizing reagent (18,19), showing that the tetracycline accumulation is an electrically neutral, ApH-dependent process. Moreover, we showed that the ApH-dependent accumulation of tetracycline occurred in liposomes made from E. coli phospholipids.* Corresponding author. MATERIALS AND METHODSBacterial strains and cell preparations. E. coli ML308-225 (3) was used in this study. Cells were grown to the mid-log phase (A610...

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

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Resource recycling and the proper treatment of animal waste to reduce its environmental impact are currently important issues for the livestock industry. A microbial fuel cell (MFC), a new type of bioreactor, is expected to play roles in both waste‐water purification and energy recovery. However, the generation of electricity from cow waste has not yet been examined. In this study, using an MFC, we examined the possibility of generating electricity from dairy‐cow waste slurry, and analyzed the properties of the treated slurry as liquid manure for resource recycling. The MFC treatment of the slurry generated electricity in a dose‐dependent manner, and the maximum power output by the MFC from a 1 g of chemical oxygen demand/L slurry was 0.34 mW/m2. After the MFC treatment, 84% of the biological oxygen demand in the slurry was removed and three essential fertilizer elements (nitrogen, phosphorus, and potassium) were retained at 84, 70, and 91% levels, respectively. The amount of ammonia nitrogen in the slurry, as an element of fast‐release fertilizer, was increased by 1.9‐fold. Although the treated slurry displayed properties that made it preferable as liquid manure, further studies to improve the electrical power output by the MFC are required for practical use.