Energetics of streptococcal growth inhibition by hydrostatic pressure.

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5' -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K(+) levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 mumol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 mumol/ml at 1 atm and 5.5 mumol/ml at 408 atm. N,N'-dicyclohexylcarbodiimide at a 10 muM concentration improved growth efficiency under pressure, as did Mg(2+) or Ca(2+) ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts

Intact etioplasts with an intactness of 85% and with a cytosolic and a mitochondrial contamination of less than 10% were isolated from 8-d-old dark-grown barley (Hordeum vulgare) leaves. These plastids contained starch equivalent to 21.5 μmol of glucose per mg protein. From various likely precursors applied to isolated etioplasts, only dihydroxyacetone phosphate (DHAP) had significant effects on metabolite levels and on the internal ATP/ADP ratio. The concentration dependence of DHAP uptake exhibited saturation characteristics with half saturation at 0.36 mm DHAP and a maximal velocity of 6.6 μmol mg⁻¹ of protein h⁻¹. The transport was significantly inhibited by inorganic phosphate, pyridoxal-5′-phosphate, and 4,4′-diisothiocyano-2,2′-stilbenedisulfonate. The rate of glucose-6-phosphate uptake was much lower and not saturable up to a concentration of 10 mm. Exogenously applied [¹⁴C]DHAP was incorporated into starch at a rate of 0.14 μmol of DHAP mg⁻¹ of protein h⁻¹. Enzyme activities required to convert DHAP into starch were found to be present in etioplasts. Furthermore, enzymes generating ATP from DHAP for ADPglucose synthesis were also detected. Finally, a scheme is presented suggesting DHAP uptake to serve both as carbon skeleton and as energy source for starch synthesis, mediated by a translocator with properties similar to those of the triose phosphate translocator from chloroplasts.     

Adenosine triphosphatase in isolated membranes of Staphylococcus aureus

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5′-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 μmole per min per mg of protein under optimal conditions. The enzyme was Mg⁺⁺-dependent and K⁺- or Na⁺-stimulated. Maximal activity was observed with a molar adenosine 5′-triphosphate (ATP) to Mg⁺⁺ ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5′-diphosphate. Inorganic pyrophosphate and the 5′-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.     

Transition from Anoxygenic to Oxygenic Photosynthesis in a Microcoleus chthonoplastes Cyanobacterial Mat

Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter⁻¹ at night and 1 atm pO₂ during the day. During experimental H₂S to O₂ transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H₂S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter⁻¹, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter⁻¹ (25 to 75 μmol of H₂S liter⁻¹). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter⁻¹ (500 μmol of H₂S liter⁻¹) in the overlying water. The implications of such a localized O₂ production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Effects of High Dissolved Inorganic and Organic Carbon Availability on the Physiology of the Hard Coral Acropora millepora from the Great Barrier Reef

Coral reefs are facing major global and local threats due to climate change-induced increases in dissolved inorganic carbon (DIC) and because of land-derived increases in organic and inorganic nutrients. Recent research revealed that high availability of labile dissolved organic carbon (DOC) negatively affects scleractinian corals. Studies on the interplay of these factors, however, are lacking, but urgently needed to understand coral reef functioning under present and near future conditions. This experimental study investigated the individual and combined effects of ambient and high DIC (pCO₂ 403 μatm/ pH_Total 8.2 and 996 μatm/pH_Total 7.8) and DOC (added as Glucose 0 and 294 μmol L^-1, background DOC concentration of 83 μmol L^-1) availability on the physiology (net and gross photosynthesis, respiration, dark and light calcification, and growth) of the scleractinian coral Acropora millepora (Ehrenberg, 1834) from the Great Barrier Reef over a 16 day interval. High DIC availability did not affect photosynthesis, respiration and light calcification, but significantly reduced dark calcification and growth by 50 and 23%, respectively. High DOC availability reduced net and gross photosynthesis by 51% and 39%, respectively, but did not affect respiration. DOC addition did not influence calcification, but significantly increased growth by 42%. Combination of high DIC and high DOC availability did not affect photosynthesis, light calcification, respiration or growth, but significantly decreased dark calcification when compared to both controls and DIC treatments. On the ecosystem level, high DIC concentrations may lead to reduced accretion and growth of reefs dominated by Acropora that under elevated DOC concentrations will likely exhibit reduced primary production rates, ultimately leading to loss of hard substrate and reef erosion. It is therefore important to consider the potential impacts of elevated DOC and DIC simultaneously to assess real world scenarios, as multiple rather than single factors influence key physiological processes in coral reefs.     

Effect of Nickelous and Other Metal Ions on the Inhibition of Rumen Bacterial Metabolism by 3-(3′-Isocyanocyclopent-2-Enylidene)Propionic Acid and Related Isocyanides

3-(3′-Isocyanocyclopent-2-enylidene)propionic acid at a concentration of 2 to 5 μg ml⁻¹ inhibited cellulose digestion by a mixed culture of rumen microorganisms and in other experiments inhibited the degradation of timothy hay (Phleum pratense) in a digestibility test. At isocyanide concentrations of 12 μg ml⁻¹ the fermentation activity of rumen fluid, measured by its dehydrogenase activity, was inhibited but not abolished. All of these isocyanide effects were reversed by the incorporation of nickelous ion into the solutions of the systems under study. The activity of 1 mol of isocyanide is reversed by about 1 mol of Ni²⁺ and, in the case of the cellulose digestion test, by about 1 mol of Co²⁺. Of some 15 other ions tested only Pd²⁺ and possibly chromium reversed the effect of the isocyanide.     

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m⁻² s⁻¹ on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell⁻¹ and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m⁻² s⁻¹, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m⁻² s⁻¹ than at 10 μmol of photons m⁻² s⁻¹ and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

Selective Inhibition by 2-Bromoethanesulfonate of Methanogenesis from Acetate in a Thermophilic Anaerobic Digestor

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58°C) anaerobic digestor were studied. It was found from short-term experiments that 1 μmol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from ¹⁴CH₃COO⁻, whereas 50 μmol/ml was required for complete inhibition of ¹⁴CO₂ reduction. When 1 μmol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 μmol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H₂, and ethanol.     

Effect of Sulfide on Nitrogen Fixation in a Stream Sediment-Water System

Nitrogen fixation (C₂H₂ reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C₂H₄ production occurring in the presence of 8 μmol of S²⁻ per ml of stream water. Sodium sulfide at concentrations of 16 μmol of S²⁻ per ml or greater inhibited nitrogen fixation, with 64 μmol of S²⁻ per ml being completely inhibitory. Sulfide at levels of 16 μmol/ml or above inhibited CO₂ production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO₂ production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 μmol of S²⁻ per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H₂, H₂S, and other sulfur species at various pH values, it appeared that H₂S was the toxic entity and that HS⁻ was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS⁻ from H₂S.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

Mode of Action of Lomofungin

Lomofungin inhibited the growth of some yeasts and mycelial fungi at concentrations between 5 and 10 μg/ml. At such concentrations, there was no decrease in endogenous and exogenous oxygen consumption, and even 50 μg of antibiotic per ml caused only slight decreases. The permeation of the cell membrane was changed so that leakage of ninhydrin-positive substances was reduced, and the uptake of ¹⁴C-labeled glucose, amino acids, uracil, and thymidine was decreased at concentrations as low as 4 μg/ml. Protein synthesis in whole cells of Saccharomyces cerevisiae was reduced 35% at 10 μg/ml. However, the antibiotic did not reduce the incorporation of phenylalanine-U-¹⁴C into polypeptides with cell-free systems of Rhizoctonia solani and S. cerevisiae. The synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) was inhibited even at concentrations of lomofungin of 4 μg/ml. Since RNA synthesis was inhibited at lower concentrations and earlier than DNA synthesis, the primary site of action of the antibiotic appears to be the synthesis of RNA.     

Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [¹⁴C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [¹⁴C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.     

Mode of Action of Lomofungin

Lomofungin inhibited the growth of some yeasts and mycelial fungi at concentrations between 5 and 10 μg/ml. At such concentrations, there was no decrease in endogenous and exogenous oxygen consumption, and even 50 μg of antibiotic per ml caused only slight decreases. The permeation of the cell membrane was changed so that leakage of ninhydrin-positive substances was reduced, and the uptake of ¹⁴C-labeled glucose, amino acids, uracil, and thymidine was decreased at concentrations as low as 4 μg/ml. Protein synthesis in whole cells of Saccharomyces cerevisiae was reduced 35% at 10 μg/ml. However, the antibiotic did not reduce the incorporation of phenylalanine-U-¹⁴C into polypeptides with cell-free systems of Rhizoctonia solani and S. cerevisiae. The synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) was inhibited even at concentrations of lomofungin of 4 μg/ml. Since RNA synthesis was inhibited at lower concentrations and earlier than DNA synthesis, the primary site of action of the antibiotic appears to be the synthesis of RNA.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Effects of Cadmium on the Growth and Uptake of Microorganisms

Six species of microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus acidophilus, Staphylococcus aureus, Streptococcus faecalis and Actinomyces niger, were grown under suitable conditions in appropriate media. Cadmium chloride was added to provide 0, 5, 10, 20, 40, and 80 μg of Cd per ml. At 40 and 80 μg of Cd per ml. E. coli and B. cereus grew well and the other species were repressed. Cd uptake patterns differed significantly among the species tested. The significance of these data with respect to Cd in food chains is discussed.     

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g⁻¹ fresh weight were found. Plants that accumulated up to 2 to 3 μmol g⁻¹ fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g⁻¹ fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g⁻¹ fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.     

Identification of Indole Derivatives as Self-Growth Inhibitors of Symbiobacterium thermophilum, a Unique Bacterium Whose Growth Depends on Coculture with a Bacillus sp.

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a Bacillus sp. Recently, we discovered that CO₂ generated by Bacillus is the major inducer for the growth of S. thermophilum; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by S. thermophilum. We succeeded in purifying two substances from an ether extract of the culture supernatant of S. thermophilum by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3′-indolyl)indoxyl (BII) and 1,1-bis(3′-indolyl)ethane (BIE). The pure growth of S. thermophilum was inhibited by authentic BII and BIE with MICs of 12 and 7 μg/ml, respectively; however, its growth in coculture with Bacillus was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 μg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MICs (<0.1 μg/ml) while a marked amount of BIE (6 to 7 μg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that Bacillus benefits S. thermophilum by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.