The biosynthesis of N-putrescinylthymine (NpT), a modified pyrimi-dine which occurs in the DNA of phage φW-14, was studied. Prior to this study, the metabolism of arginine and polyamines in the host organism, Pseudomonas acidovorans 29, was investigated. P. acidovorans transported ornithine and arginine but not putrescine. Neither amino acid was used as a sole source of nitrogen. Thus arginine cannot be catabolized to putrescine and ornithine is not catabolized to γ-aminobutyrate. Ornithine was synthesized from glutamate but the operation of this pathway was not inhibited by high concentrations of arginine. Since ornithine was decarboxylated to putrescine, this single route for the synthesis of polyamines is unusual in that ornithine biosynthesis is constitutive. P. acidovorans and φW-14 contain an unusual complement of polyamines: spermidine, putrescine and 2-hydroxyputrescine. Their respective bacterial concentrations (mM) were: 3-5, 50 and 45, phage infection led to an increase in the proportion of putrescine. ¹⁴C-Ornithine was used to label NpT in phage DNA. ¹⁴C-5-Ornithine exclusively labelled NpT, whereas ¹⁴C-1-ornithine was totally ineffective in this capacity. Hence, the carboxyl group of ornithine is not a constituent of the NpT molecule. Labelling of NpT with ¹⁴C-3-serine demonstrated a tetrahydrofolate (THF) involvement in its biosynthesis. The use of ³H-2,3-serine showed that N¹⁰methylene THF was the carbon donor for the pyrimidine precursor of NpT. It is proposed that NpT is synthesized in the following way: 5-hydroxymethyl deoxyuridine monophosphate is formed by the interaction of N¹⁰ methylene THF and deoxyuridine monophosphate; putrescine then condenses with the hydroxymethyl group of the nucleotide to yield N-putrescinyl-thymine. / Science, Faculty of / Microbiology and Immunology, Department 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 is a common cause of chronic respiratory infection in cystic fibrosis (CF). Infection is established within the lung epithelial mucus layer, through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulphated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes display mucin sulphatase activity, including P. aeruginosa. Using ion chromatography, the levels of sulphation on different respiratory mucins and the availability of inorganic sulphate in CF sputum were quantified, and the ability of clinical P. aeruginosa isolates to desulphate mucin was tested by providing mucin as a sole sulphur source for growth. All tested P. aeruginosa strains isolated from the CF lung were able to use human respiratory mucin as source of sulphur for growth, whereas other non-clinical Pseudomonas species were not. However, measured levels of inorganic sulphate in CF sputum suggest that bacteria resident in the lung have sufficient inorganic sulphate for growth and are unlikely to require access to mucin-sulphur as a sulphur source during chronic infection. This was confirmed when expression of sulphate-repressed P. aeruginosa genes, atsK and msuE, were found by quantitative PCR to be repressed in CF sputum. These results indicate that sulphate-starvation is unlikely to occur in pathogens residing in CF sputum and, therefore, mucin desulphation may have an alternative purpose in the association between P. aeruginosa and CF airways.Transcriptomic studies showed enhanced expression of 5 main islands on the P. aeruginosa genome in the presence of mucin as a sulphur source, when compared to sulphate. These islands include general sulphur-starvation response gene clusters, encoding desulphurizing enzymes AtsA, SsuD and MsuD, plus the locus PA2083-PA2094. This locus has not been characterised but encodes a putative sulphonatase, an extracellular-function (ECF) type sigma factor, with associated TonB-dependent transducer, and Major Facilitator Superfamily transporters. Transcriptional studies of this locus in response to various sulphur sources revealed that the locus comprises two transcriptional units under sulphate-limited conditions, and putative σ70-type promoters were identified using 5’-RACE and sequence alignment. Transcriptional regulation of the locus is contributed to by the encoded ECF-type σ factor and anti-σ factor, as a mutant carrying only a disrupted copy of these genes displayed a lack of transcriptional downregulation of the locus in the presence of sulphate. The influence of mucin on transcription levels of the locus was also investigated by RT-qPCR, showing that for maximum transcriptional levels both sulphate-limitation and the presence of mucin are required. However, despite repression of P. aeruginosa sulphate-regulated genes in CF sputum, the level of expression of the locus PA2083-PA2094 in CF sputum was comparable to that of P. aeruginosa culture grown in sulphate-limited conditions. The influence of the lung environment may, therefore, have a greater impact on expression levels of the locus than seen in in vitro studies with mucin. To further investigate the role of the locus, mutants were constructed and screened for changes in their ability to utilise a range of sulphur sources, including mucin, for growth. However, none of the mutants showed significant change in their growth patterns in response to any of the other sulphur sources tested, suggesting that the locus may be involved in desulphurization of a compound not tested in this study or may be functionally replaced by other organosulphur utilisation pathways in its absence. With the aim of identifying genes involved in mucin desulphurization, a P. aeruginosa transposon library was generated, combining the high-throughput nature of a random library with the variable expression reporter capabilities afforded by a promoterless GFP insert. The GFP reporter transposon produced varying fluorescence levels over time during growth of individual mutants, based on the activity of the promoter upstream of the transposon insertion site. A preliminary method was devised using fluorescence-activated cell-sorting to isolate mutants displaying altered GFP expression levels in response to sulphate availability and to mucin. Overall, this work explores the prevalence and importance of mucin desulphurization by P. aeruginosa, with relation to cystic fibrosis lungs, and provides some insight into the transcriptional patterns of the P. aeruginosa locus PA2083 to PA2094.

Intact bovine Longissimus dovsi muscle was subjected to a mild washing procedure in order to reduce the concentration of the sarcoplasmic fluid. Intact and washed muscle samples were inoculated with Pseudomonas fragi to evaluate the effect of a sarcoplasmic reduction on bacterial growth and subsequent spoilage during storage at 4°C for 12 days. Aseptic controls were stored under similar conditions. Alterations in the water-soluble, salt-soluble, urea-soluble and urea-insoluble protein fractions, as well as the total carbohydrate, pH and bacterial numbers, were monitored in both intact and washed inoculated muscle samples. Scanning and transmission electron microscopy were employed to monitor ultrastructural changes on the muscle surface as a consequence of the growth of P. fragi. Analysis of water-soluble components (non-protein nitrogen, water-soluble proteins and carbohydrates) indicated that the washing procedure effectively removed the majority of these components. Increases in the extractability of the water-soluble and salt-soluble protein fractions were observed in the intact inoculated muscle sample. Alterations in the SDS-gel electrophoretic pattern of the water-soluble, salt-soluble, urea-soluble and urea-insoluble proteins were evident. Total carbohydrate decreased as a result of growth of P. fragi. An increase in pH of the intact muscle occurred as bacterial numbers increased. Significantly (P < 0.01) higher growth rates were observed on the intact muscle tissue than the washed muscle tissue. Relatively little change in the non-protein nitrogen, water-soluble and salt-soluble protein content was observed in the washed inoculated muscle tissue. A slight decrease in total carbohydrate was seen. Minor changes in the SDS-gel electrophoretograms of the salt-soluble proteins were apparent. Little change in pH of the washed inoculated sample occurred due to the growth of P. fragi. Scanning electron micrographs indicated that surface degradation of both intact and washed inoculated muscle samples were apparent only in areas of localized colonization. Glycocalyx appeared to mediate not only cell to cell attachment, but also cell to muscle surface adhesion. Bacteria were observed growing between muscle fibers. Transmission electron micrographs of intact inoculated muscle tissue confirmed the mediation of glycocalyx in bacterial adhesion. Cellular evaginations were present on the surface of the bacteria. Autolysis was minimal in both intact and washed aseptic muscle controls. / Land and Food Systems, Faculty of / Graduate

Con el objetivo de caracterizar bioquímicamente la actividad lipolítica de Pseudoalteromonas atlantica PAR 2, aislada de la Bahía de Paracas (Ica), se procedió a cultivar la bacteria en medio LB (Luria-Bertani) a temperatura ambiente durante 24 horas. Para determinar la actividad lipolítica de la Pseudoalteromonas atlantica PAR 2, se utilizó agar SW 5 % con tributirina 1 % y se incubó a temperatura ambiente por 48 horas; la hidrólisis del sustrato se evidenció por la formación de un halo transparente. Así también, se cuantificó la actividad lipolítica utilizando como sustratos por un lado tween 80 y por otro, aceite de oliva al 1 %; los cuales no fueron degradados; esta respuesta evidenció que la enzima en estudio es una lipasa del grupo VI o esterasa. Por ese motivo, se utilizó como sustrato -nitrofenol acetato en buffer fosfato 25 mM pH 7, el producto liberado se midió por espectrofotometría a 405 m y se obtuvo una actividad enzimática de 79,32 mol/mL y una actividad específica de 661,00 mol/mg de proteína en el extracto crudo. Pseudoalteromonas atlantica PAR 2 produce una lipasa del grupo VI o esterasa, la cual además presenta actividad óptima a 20 ºC, pH 7 y concentración salina 5 %. -- Palabras clave: Pseudoalteromonas atlantica, actividad lipolítica, esterasa, -nitrofenol acetato. / -- In order to characterize biochemically the lipolytic activity of Pseudoalteromonas atlantica PAR 2, isolated from the Bay of Paracas (Ica), it was proceeded to grow the bacteria in LB medium (Luria-Bertani) at environment temperature during 24 hours. To determine lipolytic activity of Pseudoalteromonas atlantica PAR 2, SW 5 % agar with tributyrin 1 % was incubated at environment temperature for 48 hours, where substrate hydrolysis was evidenced by the formation of a transparent halo. Additionally, lipolytic activity was quantified using as substrates Tween 80 on one side and on the other, olive oil, 1%, which were not degraded, this response revealed that the enzyme under study is a lipase or esterase group VI. For that reason, was used as substrate -nitrophenol acetate in 25 mM phosphate buffer pH 7, the released product was measured by spectrophotometry at 405 m and was obtained 79,32 mol/mL as enzymatic activity, and 661,00 mol/mg protein as specific activity in the crude extract. Pseudoalteromonas atlantica PAR 2 produces a lipase group VI or esterase, which also has optimal activity at 20 °C, pH 7 and 5% salt concentration. -- Keywords: Pseudoalteromonas atlantica, lipolytic activity, esterase, -nitrophenol acetate / Tesis

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.