Abstract(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c 2 oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c 1 and c 2 , the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a lowpotential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/ mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c 1 and c 2 , and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycinsensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/ mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 be...

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd 1 -type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15-to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H202.Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg2+-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 ,uM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.The complexing agent nitrilotriacetate (NTA) is used for a range of different purposes, and one of its most controversial applications is that as a substitute for sodium triphosphate in laundry detergents (28). Many representatives of both obligately aerobic and facultatively denitrifying microorganisms which can use NTA as a sole source of nitrogen, carbon and energy have been isolated. The majority of such isolates are gram-negative, obligately aerobic rods (1,5,10,14,29) which previously have been identified as Pseudomonas spp. Recently, it has been shown that these isolates belong to a new genus for which the name "Chelatobacter" has been proposed (6).The biochemical pathway for NTA degradation was first investigated in the two virtually identical "Chelatobacter" isolates T23 (1) and ATCC 29600 (9). In both strains, a monooxygenase was reported to be responsible for the oxidative conversion of NTA (24,25). In this paper, the characterization of the NTA-Mo in cell extracts, as well as the purification, reconstitution, and characterization of a functional twocomponent NTA-Mo, is reported. MATERIALS AND METHODSGrowth of the microorganism. "Chelatobacter" strain ATCC 29600 was obtained from the American Type Culture Collection, Rockville, Md., and was maintained on a synthetic medium containing 1 g of NTA liter-' as described previously (5). In order to avoid excretion of large amounts of ammonia, the strain was grown on a mixture of NTA and acetate (1 g of each liter-') for large-scale growth (100 lit...

the derivation of improved quantitative structure-activity relationships (QSARs). Furthermore, the approach taken in this study offers the possibility to evaluate quantitatively synergistic and antagonistic effects of different phenolic compounds on energy transduction when such compounds are present in mixtures.

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Abstract-A new, mechanistically based approach is presented for the quantitative determination of the uncoupling and inhibitory activity of compounds interfering with energy transduction. Time-resolved spectroscopy of single-turnover events in the photosystem of the photosynthetic bacterium Rhodobacter sphaeroides can quantitatively distinguish uncoupling from inhibition. The decay kinetics of the membrane potential after a single turnover flash are used as a measure of uncoupling activity, and the redox kinetics of several components of the electron transfer chain are used as indicators of specific inhibition at various potential inhibitory sites. Results are presented for 21 nitrated and chlorinated phenols, some reference uncouplers, and some anisoles. Inhibition was exclusively detected at one specific quinone binding site, the quinone reductase site Q i . For most phenols, uncoupling was observed at lower concentrations than inhibition with the exception of alkylated 2,6-dinitrophenols and 2,4,6-trichlorophenol, where both effects occurred in the same concentration range. No direct correlation was observed between the uncoupling and inhibitory activity of a given compound. The data obtained with this new method correlate well with data from various bioassays on energy-transducing systems, indicating that this method may also be well suited as a screening tool for compounds suspected to interfere with energy transduction.

In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle.

Several investigations have shown that during growth in carbon-limited chemostats the simultaneous utilisation of carbon substrates which usually provoke diauxie under batch conditions, i.e., 'mixed substrate growth,' is probably the rule under ecologically relevant growth conditions. In contrast, the models presently available for the description of the kinetics of microbial growth are all based on the use of single substrates. Systematic studies in chemostat culture have shown that steady-state residual concentrations of individual compounds were consistently lower during mixed substrate growth than during growth with the single substrates. This effect is clearly demonstrated for the case of Escherichia coli growing with mixtures of glucose plus galactose. The data presented indicate that the extent of reduction of steady-state residual substrate concentration is dependent on the proportions of the substrates in the mixture, the nature of substrates mixed and the regulation pattern of enzymes involved in their breakdown. If this behaviour can be shown to be typical for growth under environmental conditions, it may provide an explanation why microbes still grow relatively fast at the low substrate concentrations encountered in nature.