Please read the abstract in the section 00front of this document / Dissertation (MSc(Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

The nature of the reserves of Pseudomonas aeruginosa that are oxidized during endogenous respiration was studied by following the changes in the chemical constituents and in the distribution of radioactivity of starving cells that had been grown on C¹⁴-labeled substrates. The total protein and nucleic acid of cell suspensions decreased during starvation. Deoxyribonucleic acid increased slightly, whereas ribonucleic acid decreased. C¹⁴O₂ was evolved from endogenously respiring cells specifically labeled in the nucleic acid fraction and from cells specifically labeled in the protein fraction. Chemical fractionation of C¹⁴-labeled cells showed a decrease in hot trichloroacetic acid-soluble and insoluble compounds, indicating that the C¹⁴O₂ arose from the degradation of RNA and protein and not from free pool compounds. A decrease in ribosomal RNA and protein was evident from physical fractionations of starved labeled cells. An enzyme responsible for the initiation of ribosomal degradation was found to be associated with the ribosome fraction and was identified as polynucleotide phosphorylase. The enzyme was inactive in high magnesium concentrations but was active under conditions which allowed the dissociation of the large ribosomal units into 50S and 30S components. Polynucleotide phosphorylase was not solubilized by the dissociation of the 70S ribosomes but remained firmly attached to the 50S subunit. The oxidation of exogenous substrates resulted in varying degrees of suppression of the oxidation of endogenous RNA and this suppression was attributed to the relative stabilizing effect that the exogenous substrates exerted on the ribosomes. The oxidation of endogenous protein was depressed during the oxidation of exogenous glucose, aspartic acid and adenosine and was increased during the oxidation of α-ketoglutaric acid. The response of endogenous respiration to the oxidation of exogenous substrates appeared to be related to a requirement for ammonium ions for assimilation of carbon. / Land and Food Systems, Faculty of / Graduate

The non-phosphorylated oxidative pathway of glucose dissimilation has been established in Pseudomonas aeruginosa and evidence for phosphorylated pathways, other than the Embden-Meyerhof scheme, has been obtained. In the present study the non-phosphorylated and phosphorylated pathways of glucose degradation have been investigated with cell-free extracts of this organism. Gluconolactone was shown to be an intermediate in the oxidation of glucose to gluconic acid. The enzymatic hydrolysis of the lactone ring has an absolute magnesium ion, or divalent cation requirement. In the presence of phosphate buffer magnesium was chelated and effectively removed from participation in the enzymatic reaction. As has been reported in the literature, the product of glucose and gluconic acid oxidation was identified as 2-ketogluconate. In the presence of adenosine triphosphate (ATP), glucose and gluconate are phosphorylated and the kinases involved, therefore, link the non-phosphorylated with the phosphorylated pathways. The demonstration of triphosphopyridine nucleotide (TPN) linked dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, as well as the production of glucose-6-phosphate and 3-phosphoglyceraldehyde from cell-free extracts with gluconate or ribose plus ATP, illustrated the presence of a functional pentose phosphate cycle in this organism. An active 6-phosphogluconate dehydrase and a 2-keto-3-deoxy-6-phosphogluconate aldolase were demonstrated by the production of pyruvic acid from 6-phosphogluconate and indicated the presence of the Entner-Doudoroff pathway. The oxidation of 3-phosphoglyceraldehyde to 3-phosphoglyceric acid initiated by a TPN specific 3-phosphoglyceraldehyde dehydrogenase, and the conversion of phospho-enol-pyruvate to pyruvic acid was shown. It is suggested that the trioses are immediately concerned in the observed CO₂ fixation by this organism. Fructose-1,6-diphosphate aldolase, fructose-1,6-diphosphate phosphatase and phosphohexoisomerase may be involved in the formation of glucose-6-phosphate from triose phosphates. A direct link between 2-ketogluconate and the phosphorylated pathways could not be shown but the reduction of the phosphate ester of the compound was demonstrated. The feasibility of 2-ketogluconate undergoing a 3:3 split is presented. No attempt has been made to estimate the relative importance of the various pathways of glucose dissimilation as it is felt that this is determined by the conditions and stages of growth of the organism. / Land and Food Systems, Faculty of / Graduate

An effort has been made to demonstrate that the major pathway for glucose oxidation in Pseudomonas aeruginosa (ATCC 9027) involves the sequence of reactions: glucose →gluconate → 2-ketogluconate → 2-keto-6- phosphogluconate → 6-phosphogluconate. It appears however, that extracts of this organism are capable of phosphorylating glucose directly, that is, to yield glucose-6-phosphate and subsequently 6-phosphogluconate. A study of this latter pathway was felt to be necessary in order to evaluate the likelihood of it being a major alternative to the established non-phosphorylated pathway. Since it is known that glucose-6-phosphate dehydrogenase from P. aeruginosa and other microorganisms as well as from certain animal tissues exhibits a marked sensitivity to various nucleotides particularly to adenosine triphosphate. A study of this inhibition was made in order to assess the possible role that this sensitivity may play in determining the importance of this pathway as the major route of glucose oxidation. Enzyme fractionation studies revealed that hexokinase and glucose-6-phosphate dehydrogenase could be separated either by an ethanol fractionation or by an alkaline ammonium sulfate fractionation. The best separation of dehydrogenase was obtained using ethanol although hexokinase could only be isolated using the alkaline ammonium sulfate method. Cell free extracts of P. aeruginosa oxidize glucose to 2-keto-gluconate but carry the reaction no further. This represents a consumption of l µM of oxygen per µM glucose. In the presence of ATP the amount of oxygen consumed was reduced to a maximum of 0.5 µM per µM glucose, indicating the accumulation of a compound less oxidized than 2-ketogluconic acid. 6-phosphogluconate appeared to conform to the requirements of such a compound. Chromatographic analysis of reaction mixtures containing ATP revealed the accumulation of a phosphorylated compound which could not be identified. Under in vitro conditions both pathways appear to be operable but the non-phosphorylated pathway accounts for most of the glucose in the metabalizing organism. / Land and Food Systems, Faculty of / Graduate

Isolated membranes of Pseudomonas aeruginosa were found to bind radioactive isoleucine and proline, two amino acids for which active transport systems are known. The active transport systems in whole cells of this organism are energy dependent; the binding systems in isolated membrane preparations are not energy dependent, but are inducible, require magnesium ions and are stable to short periods of sonication usually sufficient to destroy whole cells. An assay measuring the binding of radioactive amino acid to amino acid binding protein present in isolated membrane preparations of P. aeruginosa was developed and discussed. Cells induced to high levels of amino acid transport produced equivalent levels of binding on isolation of membranes from these cells. Cells repressed for amino acid transport did not lose a corresponding level of binding on isolation of their membranes, suggesting involvement of more than one protein in the active transport system of that particular amino acid. Evidence was found to substantiate claims that active transport systems are family specific, however it was also determined that the aliphatic amino acid binding system was not stereospecific. Isolated membrane preparations of P. aeruginosa were found to produce adenosine triphosphate, but this energy-rich, phosphate bond compound did not appear to function in binding of radioactive amino acid to membrane preparations. Its possible functions are discussed. Several methods of isolation of proteins with binding properties from isolated membranes and from osmotic shock supernatant fluids were attempted and discussed. There were indications of a proline binding protein present, but no evidence of an isoleucine binding protein, in the osmotic shock supernatant fluid. The isoleucine binding-protein, or proteins, appeared as an integral part of the cytoplasmic membrane. The data were discussed in an attempt to clarify the mechanism of amino acid transport in P. aeruginosa and to define the role of the amino acid binding proteins in the phenomenon of active transport. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

An aldehyde dehydrogenase was found in cell extracts of Pseudomonas aeruginosa ATCC 9027 grown on several carbon sources. It was present in highest concentration in cell extracts after growth of the organism on ethylene glycol or ethanol . The enzyme from ethanol-grown cells was purified by protamine sulfate, ammonium sulfate, acetone, and isoelectric precipitation, ion exchange chromatography and gel filtration. After an eighteen-to twenty-fold purification with a twenty-three per cent yield of activity a homogeneous preparation was obtained, as evidenced by ultracentrifugation, electrophoresis, and other criteria. The enzyme was found to be unstable in crude preparations. This instability was overcome by the use of bisulfite buffer. The enzyme oxidizes a wide variety of aldehydes. The products of glycolaldehyde and glyceraldehyde oxidation were identified as the free acids. The pH optimum for the reaction was found to be between pH 8.0 and 8.6. The enzyme is more active with NAD⁺ as the hydrogen acceptor than with NADP⁺. Potassium or ammonium was found to be essential for activity. Less activity was obtained in the presence of rubidium. Aldehyde dehydrogenases from five other species of Pseudomonas were also activated by potassium. Michaelis constants for aldehyde substrates, NAD⁺, NADP⁺, and the activating ions were determined. In addition to the activating ion, a reducing agent was required for enzymatic activity. It could be replaced, in part, by EDTA or o-phenanthroline. No inhibition was observed with EDTA, but o-phen-anthroline inhibited the enzyme reaction in the presence of a reducing agent. However, zinc was not found to be present in the purified aldehyde dehydrogenase. Aldehyde dehydrogenase also was inhibited by iodoacetamide, iodoacetate, arsenite,Cu⁺⁺ , and p-chloromercuribenzoate. Enzymatic activity also was lost when trypsin was added to the enzyme preparation. This loss of activity and the inhibition by the alkylating agents were specifically prevented by the addition of the activating ion and NAD⁺ to the enzyme preparation. Some protection from digestion by trypsin was afforded by potassium alone. However, in the absence of NAD⁺ potassium accelerated the rate of inhibition by alkylating agents. A molecular weight of 200,000 was determined for aldehyde dehydrogenase by several methods. At low ionic strength the enzyme underwent a partial dissociation with loss of enzymatic activity. This dissociation could be reversed by increasing the salt concentration. Dissociation and association of the enzyme into subunits of approximately equal size could be followed in the ultracentrifuge and on starch gel electrophoresis. The dissociated form of the enzyme was isolated after starch gel electrophoresis and found to be completely inactive. Full enzymatic activity was obtained only when the associated enzyme was protected from oxidation. The enzyme was soluble below its isoelectric point (pH 4.8) but denatured as evidenced by sedimentation, diffusion, and viscosity studies. The molecular weight of the enzyme preparation at pH 3.0 was estimated to be approximately one-half of that found at pH 7.0.Aldehyde dehydrogenase contained relatively large amounts of all common amino acids. The lowest amount was obtained for cysteic acid: 23 to 24 residues per mole. Studies with ¹⁴C-iodoacetamide showed that the enzyme was completely inhibited when approximately three moles of iodoacetamide were taken up per mole of enzyme. Following chymotryptic digestion of labelled aldehyde dehydrogenase, a fraction containing a large percentage of the radioactivity was partially purified by ion exchange chromatography and gel filtration. This fraction contained one peptide species, or several very similar peptide species, probably derived from the active site of the enzyme. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The structure ribosomes of Pseudomonas aeruginosa and Escherichia coli were compared. The thermal denaturation profiles of the P. aeruginosa ribosomes were consistently at higher temperatures than the corresponding profiles for those of E. coli. The differences in the thermal denaturation profiles of the ribosomal RNAs were not as pronounced. Although the numbers of the proteins resolved from the ribosomal subunits of E. coli and P. aeruginosa were approximately the same, the distribution of these proteins in the gel was markedly different. The optimum conditions for the resynthesis of ribosomes in P. aeruginosa have been established. It was demonstrated that the resynthesis of ribosomes proceeds through a number of ribosomal precursors. By isotope labelling experiments the rate of labelling of the RNA moiety of the ribosome was found to be different from the rate of labelling of the protein moiety. Furthermore both labels were found to accumulate in the ribosomes at the expense of the ribosomal precursors. The following precursors were resolved by sucrose density gradient centrifugation: 16S → 20S →23S → 25S →28S→30S → 32S → 34S → 40S → 43S → 50S The 16S to 28S precursors were not characterized well enough to be placed either in the 50S or 30S sequence. There was evidence that the ribosomal RNA itself had precursors, 9S and 12S. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Pseudomonas aeruginosa has been shown to actively transport and accumulate twenty common amino acids by systems with enzymatic properties; that is the systems are energy dependent, temperature sensitive, are saturated at high amino acid concentrations and are lost by mutation. During growth on a synthetic, amino acid free medium this microorganism maintained a low, but significantly concentrated heterogeneous pool of amino acids for syntheses and this pool (native pool) was found to be in equilibrium with low levels of exogenous amino acids with at least one exception. Amino acid pools established from an exogenous source were found to behave differently. Whereas some amino acids were unchanged during the passage through the intracellular pool others underwent extensive degradation. Some amino acids or their degradation products were shown to be compartmentalized or made unavailable for metabolism. Proline did not form large pools under physiological conditions due to an imbalance between the rate of transport and the rate of protein synthesis. A multiplicity of intracellular proline pools was elucidated by inhibitors and studies at low temperatures. The amino acid transport systems operative at very low exogenous amino acid concentrations were shown to be strongly stereospecific. Several transport systems were elucidated by competitive inhibition studies and were found to recognize amino acids with similar chemical properties. Also very specific amino acid transport systems were demonstrated within the aromatic and basic amino acid families. The multiplicity of amino acid carrier functions was confirmed by pool displacement studies and by the selection of appropriate transport negative (Tr¯ ) mutants. Low affinity amino acid permeases or carriers were shown to operate at high amino acid concentrations for most of the amino acids tested. Low and high affinity permeases could be separately identified by kinetic studies. Amino acid transport was found to be induced to high levels by growth in the presence of the appropriate amino acid. Some evidence was presented to suggest that the control is coordinately linked to amino acid degradative enzymes. The constitutive levels of amino acid degradative enzymes were found to be lowered in the presence of glucose. With the exception of arginine, constitutive deaminases were inhibited by inorganic ammonia, whereas for the most part the constitutive transport functions were not changed. Induced transport levels were not markedly influenced by the presence of these nutrients. A novel mechanism for the transport and accumulation of amino acids was formulated. This mechanism provides for the accumulation of high and low intracellular amino acid pools by an energy dependent mechanism. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The non-phosphorylated oxidative pathway of glucose dissimilation has been confirmed in Pseudomonas aeruginosa using whole cells and cell-free extracts. The oxidation of glucose to 2-ketogluconate was complete and stoichiometric in cell-free extracts and cell-free extracts of glucose grown cells were shown to be incapable of metabolizing 2-ketogluconate. It was shown that whole cells completely degraded 2-ketogluconate and quantitatively accumulated pyruvic acid in the presence of specific inhibitors. The initial step involved in 2-ketogluconate dissimilation was found to be exceptionally labile to the effects of a variety of metabolic inhibitors. The metabolism of 2-ketogluconate was demonstrated to involve the initial phosphorylation with adenosine triphosphate (ATP) as the phosphate donor. The resultant intermediate, 2-keto-6-phosphogluconate, was identified and was shown to undergo reduction by a nicotinamide adenine dinucleotide phosphate linked reductase to 6-phosphogluconate which, in turn, was metabolized to pyruvate by enzymes of the Entner-Doudoroff pathway. Radioactivity from 2-ketogluconate-C¹⁴ was rapidly incorporated into cellular constituents, primarily protein, by washed cell suspensions of P. aeruginosa, but oxidation of 2-ketogluconate did not involve the accumulation of keto-acid intermediates. The role of 2-ketogluconic acid as a key intermediate for the conservation of excess carbon under conditions where nitrogen is limiting was discussed. / Land and Food Systems, Faculty of / Graduate

Pseudomonas aeruginosa Ps-1C produces an extracellular proteolytic enzyme which from preliminary studies appears to be inducible, and responsible for corneal destruction in injured dyes. In the present study the nutritional requirements for this bacterium to produce the proteolytic enzyme(s) were investigated. Preliminary studies indicated that proteose peptone offered the required nutrients for good enzyme production. The separation of the components of proteose peptone by Sephadex C-10 and Sephadex G-75 descending column chromatography was undertaken to illucidate the nutritional requirements. It was also noted that casamino acids hydrolysate served as a good substrate for Pseudomonas aeruginosa to produce this enzyme. The separation of amino acid groups was undertaken using paper and Ceon electrophoresis and various types of thin layer chromatography. The three amino acids found to be required for good protease production were, phenylalanine, isoleucine, and valine in their respective concentrations of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml. Isoleucine was found to be inhibiting at higher concentrations. Dextrose also inhibited protease production, but not growth, at concentrations greater than 0.05%. Divalent metal ions in varying concentrations were tested as nutritional requirements for enzyme production. Magnesium ion provided very good enzymatic activity at a concentration of 0.01 M, whereas cobalt, copper, calcium and zinc ions did not allow appreciable enzyme activity and even in some cases were inhibitive.