Duisburg, Essen, Universiẗat Duisburg-Essen, Diss., 2003.

Bochum, Universiẗat, Diss., 2001.

Hannover, Universiẗat, Diss., 2001.

Contrary to some reports in the literature Pseudomonas aeruginosa ATCC 9027 as well as other strains of Pseudomonads produced definitely larger amounts of protease when supplied with proteinaceous nutrients than in a glucose mineral salts medium. The enzyme appeared to be extracellular in character and liberated under conditions of at least partial starvation in the presence of an inducer. Of the properties of the enzyme temperature and pH optima were found to be 60°C and pH 8.0 respectively. Chelating agents were found to inhibit enzyme activity. / Land and Food Systems, Faculty of / Graduate

The earlier failure to demonstrate substrate-dependent oxidative phosphorylation in cell free extracts of Pseudomonas aeruginosa led to an investigation of the conditions affecting the incorporation of radioactive phosphorus into resting cell suspensions of this organism. Incorporation of radioactive phosphorus was shown to be dependent on the substrate concentration, the presence of magnesium ions, a source of available nitrogen and to be associated with the oxidative enzymes of the cell. The more common methods of cell breakage employed for the preparation of bacterial cell free extracts were considered detrimental to the mechanisms of oxidative phosphorylation and were abandoned in favor of a method involving the osmotic lysis of spheroplasts with versene and lysozyme. These preparations were shown to be easily separated into membranes, cytoplasm, and ribosomes by differential centrifugation and had the advantage of not having been subjected to severe physical treatments. Previous studies with cell free extracts had demonstrated the formation of ATP³² in the presence of ADP and P³², presumably through a coupled oxidative phosphorylation process. The formation of ATP³² was shown however to be the result of a coupled enzyme reaction involving polynucleotide phosphorylase and adenylate kinase (equations 1, 2 and 3), and although influenced by a concurrent oxidative phosphorylation process was itself not a measure of oxidative phosphorylation. [Equations omitted] The enzyme mediating the exchange reaction (equation 1) was shown to be polynucleotide phosphorylase and that at least in this organism this enzyme is associated with the ribosomal fraction of the cell. Oxidative phosphorylation was demonstrated in crude cell extracts prepared from succinate-grown cultures by the lysozyme-versene treatment. Maximum P:0 ratios of 2.0 with succinate as substrate and 4.3 for -keto-glutarate were obtained thus presenting further evidence for the similarity of this fundamental process in bacterial and animal tissue / Land and Food Systems, Faculty of / Graduate

A study of the endogenous respiration of the aerobic bacterium Pseudomonas aeruginosa was undertaken with a view to establishing the nature of the endogenous substrate and the relationship, if any, of the endogenous respiration to the oxidation of exogenous substrate. It was shown that the only end-products accumulating during endogenous respiration were ammonia and carbon dioxide. There were no detectable changes in the carbohydrate, lipid, nucleic acid or protein content of the cells during endogenous respiration. Inhibitor studies showed that protein was an endogenous substrate, and this was confirmed by the fact that after endogenous respiration succinate-grown cells required a slight induction period for succinate oxidation. Since keto-acids did not accumulate during endogenous respiration, the amino acids produced by protein degradation were probably oxidized to completion. Calculations based on this assumption showed that the ammonia production could account for all of the oxygen consumed. In the presence of an oxidizable substrate there was no production of ammonia. Manometric data showed that the endogenous respiration was not suppressed during the oxidation of an exogenous substrate. Inhibitor studies showed that oxidative assimilation involved the reassimilation of the ammonia produced by the endogenous respiration. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In attempting to study transamination in Pseudomonas aeruginosa, with the object of determining the range of compounds concerned and whether or not more than one enzyme is involved, an accurate, rapid, and generally applicable quantitative procedure for measuring amino acids was necessary. A method involving paper chromatography of reaction mixtures, spraying with ninhydrin and colorimetric measurement of the eluted spots was found and suitably modified. The reaction mixture was amino acid, keto acid, pyridoxal phosphate, water, phosphate buffer and enzyme. The range of activity of the crude cell-free extract was investigated by testing its ability to transaminate from 23 amino compounds to glyoxylate, α-ketoglutarate, oxalacetate and pyruvate. No transamination with pyruvate was observed and very little oxalacetate. The range of transamination with glyoxylate and α-ketoglutarate was extensive. In order to test whether or not these activities were due to one enzyme, purification was attempted. Isoleucine-glutamate was the system whose activity was followed. Partial purification of the enzyme catalyzing this reaction was achieved by precipitating the nucleic acids with protamine sulphate and subsequently fractionating with ammonium sulphate. The isoleucine-glutamate activity was most concentrated in the 50/60 fraction. Further purification of this enzyme system was attempted with the use of calcium phosphate gel adsorption and elution; ion-exchange resin columns; paper electrophoresis in phosphate buffers and ammonium sulphate elution from a celite column - all without success. Having achieved some purification of the isoleucine-glutamate catalyzing system, the range and specificity of this partially purified fraction was compared with that of crude cell-free extract. The results showed that the partially purified fraction retained the broad range of glyoxylate and α-ketoglutarate activities while the range of oxalacetate activity was greatly increased. The possibility of chemical transamination under reaction conditions was examined and it was observed that glyxoylate can be chemically aminated in every case where it was thought that enzymatic transamination might occur. The concentration of other transaminating activities in a number of ammonium sulphate fractions was examined. The systems studied were isoleucine, methionine and phenylalanine, each with α-ketoglutarate, as well as isoleucine phenylanaline and glutamate each with oxalacetate. Results indicated that the activities involving α-ketoglutarate were concentrating in the 50/60 fraction while those involving oxalacetate were concentrating in the 60/70 fraction. Specific activities corroborated these observations to a large extent. These results indicated at least two transaminases in P. aeruginosa. The glyoxylate system was re-examined by quantitative comparison of chemical and enzymatic transamination and also by stopping the reactions with trichloracetic acid rather than by heat. Each of these procedures indicated that glutamate will enzymatically transaminate with glyoxylate to form glycine. Other amino acids tested were inactive. The question of pyruvate participation was investigated and the presence of a glutamate-alanine system was found in fresh, crude preparations. This activity was not shown to occur in the 50/60 fraction. The observed facts therefore suggest the possibility of at least three transaminating systems in P. aeruginosa. / Land and Food Systems, Faculty of / Graduate

The conventional tricarboxylic acid cycle is generally accepted as the sole means of terminal respiration in aerobic micro-organisms. Cell-free extracts of Pseudomonas aeruginosa were found to contain the condensing enzyme and were able to oxidize all the intermediates of the conventional tricarboxylic acid cycle. In spite of this evidence in favour of the conventional tricarboxylic acid cycle some deviations from the normal scheme were found. Even though an ability to oxidize isocitrate was noted the cell-free extracts had no ability to equilibrate isocitrate with citrate, indicating possession of an impaired aconitase system. Furthermore, when citrate was used as substrate all attempts to isolate alpha-Ice toglutarate in the fermentation liquor failed. Moreover, the 2,4, dinitrophenylhydra-zone of glyoxylate was easily isolated in relatively large quantities when either citrate or cis-aconitate were used as substrates. Although glyoxylate was never isolated when isocitrate was used as substrate it was produced from citrate or cis-aconitate under either aerobic or anaerobic conditions. Since the reaction proceeded in the presence or absence of oxygen it was assumed to be a hydrolytic cleavage of cis-aconitate. In addition to glyoxylate, succinate was found as a product of the anaerobic degradation of citrate or cis-aconitate and in the presence of the cell- extract citrate was readily formed by synthe-i sis from glyoxylate and succinates Succinate was then shown to be oxidized by P.aeruginosa through fumarate and 1-malate to oxalacetate, indicating a similarity to the tricarboxylic acid cycle. These results represent a deviation from the conventional tricarboxylic acid cycle and show that the fragmentary evidence normally accepted is not sufficient to prove the presence of a conventional tricarboxylic acid cycle. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

During the investigation of the effect of age upon endogenous metabolism, advanced stationary phase cultures of Pseudomonas aeruginosa were found to be susceptible to cold-shock. The phenomenon was apparent through an increased oxygen uptake and an initial absence of extracellular ammonia during subsequent respiration at 30 C, which were shown to be due to the presence of an oxidizable substrate in the suspending fluid. Intracellular enzymes were released following the exposure of these cells to the cold, and a partial protection against damage was afforded by the addition of magnesium ions to the washing and suspending buffer. The storage of a reserve material for utilization during endogenous metabolism could not be demonstrated in cells grown for various periods of time in a chemically-defined medium which contained glucose in excess of that required for growth. Further, when not previously exposed to the cold, an immediate evolution of ammonia was observed when this organism was respired at 30 C irrespective of the medium in which it was cultured or the age at which it was harvested. The ribosome complement was seen to diminish during the prolonged incubation of cultures grown in the chemically-defined media, and was found to disappear almost completely when 48 hr cells, harvested from defined or complete media, were respired at 30 C for a further 48 hr. Chemical fractionation during the respiration period revealed an increase in the concentration of deoxyribonucleic acid and a decrease in the concentrations of ribonucleic acid and protein. Glucosamine was not found to be a major metabolite in the endogenous respiration of this organism. Progressive starvation resulted in a reduction in the constituitive enzymes and/or cofactors involved in the oxidation of glucose, and an ability to adapt to, and oxidize α-ketoglutarate was evident after a period of starvation which had reduced the ribosome complement to negligible proportions. Endogenously produced ammonia and acid-soluble UV-absorbing material were reincorporated upon the addition of an exogenous substrate following respiration for 48 hr, and a concurrent increase was recorded in the concentration of 50S ribosomes. / Land and Food Systems, Faculty of / Graduate

Pseudomonas aeruginosa was shown to constitutively degrade arginine via the arginine dihydrolase pathway to ornithine, which was converted both to glutamate and to putrescine. The conversion of ornithine to glutamate appeared to be the major route of arginine degradation in this organism, and was induced to higher activity after growth of the cells with arginine as the sole source of carbon and nitrogen. P. aeruginosa did not further degrade putrescine constitutively. However, growth of the cells in arginine resulted in a partial induction of succinic semialdehyde dehydrogenase, an enzyme functioning in putrescine degradation. The anabolic ornithine transcarbamylase of P. aeruginosa was repressed after growth of the organism in the presence of arginine. Pseudomonas putida and Pseudomonas fluorescens also possessed the ability to constitutively convert arginine to putrescine via the intermediates, citrulline and ornithine. However, these organisms did not oxidize arginine to the same extent as did P. aeruginosa. P. aeruginosa grew in a mixture of glucose and arginine in the presence of ammonium ions without exhibiting a diauxie effect. Glucose and arginine were oxidized concomitantly when supplied as a mixed substrate, by both growing cells and resting cell suspensions. However, assimilation studies showed that the two substrates were used to serve somewhat different biosynthetic needs. Growth of P. aeruginosa in arginine caused an increase in the rates of transport of arginine, lysine, ornithine and citrulline. Kinetic studies of arginine uptake demonstrated the presence of two uptake systems with different affinities for arginine. Inhibition studies indicated that arginine was transported by two uptake systems: a permease specific for arginine, and, with a lower affinity, for ornithine; and a general permease, which transported all the basic amino acids. Polyamines appeared to be transported by an uptake system which was induced to higher levels after growth of the cells with either arginine or putrescine as the sole source of carbon and nitrogen. P. aeruginosa was found to maintain a stable pool of putrescine when supplied with exogenous ¹⁴C-arginine or ¹⁴C-putrescine, even when the organism had previously been induced to degrade these substrates. A physical fractionation of the cells indicated that the major portion of this pool was located in the soluble cytoplasm. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate