Bruce C. Kelley

A bioluminescence method for determining adenosine 3′-phosphate 5′-phosphate (PAP) and adenosine 3′-phosphate 5′-sulfatophosphate (PAPS) in biological materials

Abstract A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis , which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence. Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.

The uptake and assimilation of sulphate by Thiobacillus ferrooxidans

Sulphate was rapidly bound by cell suspensions of Thiobacillus ferrooxidans. The binding was depressed by tetrathionate but was unaffected by Group VI anions, cysteine or methionine. Increasing uptake of sulphate was observed in cell suspensions incubated in the presence of ferrous iron. The bulk of 35S-sulphate was removed from the organisms by washing with dilute sulphuric acid and the remaining label was incorporated into cold trichloroacetic acid-soluble compounds. 35S-labelled adenosine 5′-sulphatophosphate was produced from ATP and 35S-sulphate by cell suspensions and in cell-free extracts. There was no evidence for the production of adenosine 3′-phosphate 5′-sulphatophosphate assayed by a very sensitive bioluminescence method.

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N2, no nitrogenase activity could be detected unless α-ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH4+. In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C2H2 reduced · min-1 · mg-1 dry weight and 50 nmol methylene blue reduced · min-1 · mg-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H2 produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H2 stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O2 in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O2), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.

Inhibition of nitrogenase activity by metronidazole in rhodopseudomonas capsulata.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH4+. Light-dependent C2H2 reduction and H2 production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H2-palladised asbestos prior to assay, it only partially restricted C2H2 reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H2-dependent photoreduction of 14CO2; (c) γ-glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor).Metronidazole (1 mM) completely inhibited C2H2 reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C2H2 reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.

Contribution of dissolved dinitrogen in culture solutions to growth of Rhodopseudomonas capsulata with various sources of combined nitrogen

Fixation of dissolved dinitrogen in culture solutions by the photosynthetic bacterium Rhodopseudomonas capsulata, strain B10, reduced the lag phase associated with growth with glutamate. A comparable effect was not observed with ammonium chloride. This strain assimilated nitrate but nitrogen fixation was depressed during early growth on nitrate. It is shown that nitrite, the first product of nitrate assimilation, inhibits nitrogen fixation during the early stages of cell growth.

Oxygen inhibition of the photoassimilation of CO2 in Rhodopseudomonas capsulata

The photoassimilation of 14CO2 by washed cells of the photosynthetic bacterium Rhodopseudomonas capsulata was greatly inhibited in air. The inhibition was partially reversed by either sparging with argon or by adding inhibitors, e.g. CO [50% (v/v) in air] and NaN3 (0.2 mM), which at these concentrations effectively restricted respiration. The effect of oxygen on the photoassimilation of 14CO2 may be associated with a change in the redox state of the cells resulting in less reducing equivalents being available for this process.

Utilization of 35S-thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans.

Differentially labelled 35S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated Kmvalue of 0.5 mM for 35S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound 35S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-35S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-35S]thiosulphate or 35S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.

The effects of carbon dioxide on nitrogenase-related activities of Rhodopseudomonas capsulata.

N2 fixation was investigated in suspensions of washed cells of the photosynthetic bacterium Rhodopseudomonas capsulata grown on various carbon and nitrogen sources. Maximum nitrogenase activities were common to cultures supplied with glutamate. Cells grown with nitrate contained appreciable nitrogenase activity and produced H2 in the light. Nitrogenase activity in washed cells derived from various nutritional conditions was markedly increased when NaHCO3 was included in the reaction mixtures.