Meat Juice Medium (MJM), an aqueous extract of meat, was inoculated with Pseudomonas aeruginosa and incubated for 7 d at 4$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). Two predominant compounds produced during spoilage of MJM were detected using HPLC. These compounds with retention times (RT) of 21.48 and 32.04 min were tentatively identified as acetic and butyric acids, respectively. These compounds were also produced when MJM was replaced with Brain Heart Infusion Broth medium. In later experiments, the effect of glucose supplementation on the rate of MJM spoilage was examined. Glucose 0.5% (wt/vol) was added to the MJM, inoculated with P. aeruginosa and incubated at 30$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). HPLC of samples after 1d of incubation indicated the presence of 8 predominant compounds including acetic and butyric acids. Their concentrations were, in general, higher in control samples of MJM without added glucose. Using HPLC, TLC, Pyrolysis/GC/MS, FTIR and GC-MS methodologies, the compounds with RT of 8.91, 9.67, 11.96, 13.33, 17.74, 21.48, 26.07 and 32.04 min were tentatively identified as cadaverine, 2-keto gluconic acid, fructose, lactic acid, acetic acid, methanol and butyric acid. In contrast to the results of previous researchers, cadaverine was produced in large amounts while no putrescine was produced by P. aeruginosa. During the spoilage period, the levels of glucose, fructose and total carbohydrate were monitored. Addition of glucose to MJM delayed slime production by 4 days and increase to maximum pH of 8.3 by 7 days. Results suggest that addition of glucose to MJM delays spoilage by P. aeruginosa.

Pseudomonas fragi CRDA 037 was used as source of intracellular esterase, which remained attached to the cell membrane, and characterized. Several mechanical methods of disruption were used including glass beads (MSK), sonication, French press and combinations of these methods. The cellular debris were also treated with detergents such as CHAPS and Triton X-100 in the presence of chelating agent ethylenediaminetetra acetic acid (EDTA). The esterase activity remained in the cellular debris which was therefore use as a source of enzyme for kinetic studies. In the case of glass beads homogenization, the activity was found to decrease as a function of time of disruption. The results of chemical treatment showed that the esterase was characterized in terms of detergent and EDA action as well as substrate specificity. Triton X-100 and EDTA had no effect on the esterase activity and did not denature the enzyme. The substrate specificity with cells and cellular debris were carried out. The valeric acid was the best in term of esterase activity among fatty acids used in the study. (Abstract shortened by UMI.)

Pseudomonas fragi CRDA 037 was used to produce both intracellular and extracellular lipases. The crude lipase fractions were fractionated using ammonium sulfate to obtain partially purified intracellular and extracellular lipases; the fraction precipitated at 20-60% of saturation of the intracellular proteins was retained as the source of the endolipase, whereas the culture medium precipitated at 20-40% of saturation, was used as the source of exolipase. Native gel electrophoresis (PAGE) suggested that the partially purified extracellular lipase has a molecular weight of 25,500 Da. Three major electrophoretic bands were present in the intracellular lipase fraction at 70,000, 49,000, and 35,500 Da. The partially purified lipases were characterized with respect to their optimum pH and temperature for lipase activity, and well as for their kinetics, specificities, and reactions toward inhibitors. The optimum pH for the activity of the endolipase was found to be 9.0 whereas that of the exolipase was 8.75. With respect to optimum temperature, 30$ sp circ$C was determined to be the best for the endolipase while 35$ sp circ$C was the optimal value for the exolipase. Enzyme specificity was carried out using triacetin, tributyrin, trimyristin, and triolein as substrates. The results for the exolipase indicated that the lowest K$ sb{m}$ was obtained with trimyristin and the highest K$ sb{m}$ was obtained with triolein whereas for the endolipase, the highest K$ sb{m}$ was obtained with tributyrin and the lowest was with trimyristin. Experiments carried out with inhibitors indicated that both lipases are serine lipases, sulfydryl enzymes, and that tryptophan is essential for maintaining the conformation of the proteins. The most potent inhibitor was ferrous chloride, and sodium deoxycholate was a weak inhibitor for both lipases, however, it was an activator at low concentrations for the exolipase.

Pseudomonas aeruginosa grew in the presence of 10 $ mu$g/ml tetracycline (TC) or chlorotetracycline (CTC) in a minimal medium containing Mg$ sp{++}$. Growth is inhibited, with a six-fold increase in length of the lag phase. Cells revert to sensitivity when returned to antibiotic-free medium. Substitution of Mg$ sp{++}$ in the growth medium of CTC-resistant strains with Ca$ sp{++}$ and Sr$ sp{++}$ resulted in dramatic changes in growth and cell mass of cultures. Exposure of CTC-grown cells to EDTA did not result in cell lysis. SDS-PAGE of outer membrane proteins of resistant cells revealed loss of a protein band of molecular weight 73,500 D and the appearance of a 54,000 D protein band. Growth of cells resistant to CTC was hampered by subsequent exposure to penicillin G. Chelation of divalent cations from the outer membrane of sensitive cells leading to cell disruption is postulated as the primary mode of action of this antibiotic against P. aeruginosa.

Pseudomonas chlororaphis PA23 is a promising biological control candidate against Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot in canola. A library of transposon mutants was previously created to understand the molecular mechanisms underlying the antifungal capabilities of PA23. A novel LysR-type transcriptional regulator, called PtrA, was identified as a key global regulator involved in secondary metabolite production. The function of PtrA at the molecular level was investigated in this thesis. Solubility problems encountered during the purification of PtrA redirected efforts to studying a truncated version of the protein instead. A two-step purification of the truncated protein, involving streptomycin sulfate precipitation and immobilized metal-ion affinity chromatography, yielded a highly pure protein. Preliminary crystal growth was achieved for the effector binding domain portion of PtrA. Transcriptional fusions suggested that essential regulatory binding sites of ptrA may lie somewhere between 52 and 198 bp upstream of the translational start site. The research presented in this thesis will help guide future functional studies on PtrA.

The partially purified extracellular lipase from Pseudomonas fragi CRDA 037, obtained by ammonium sulfate precipitation, was subjected to further purification by successive ion-exchange and size-exclusion chromatographies using the Fast Protein Liquid Chromatography system. The purification of the partially purified lipase resulted in two enzymatic fractions, FIVa ' and FIVb', with a purification-fold of 169 and 195, respectively. Native electrophoretic analyses revealed the presence of three bands for fraction FIVa', with estimated molecular weights (MW) of 16.2, 25.8 and 38.5 kDa and two bands for FIVb' , with estimated MW of 15.2 and 25.8 kDa. The two purified fractions, FIVa' and FIVb', showed an optimum pH of 9.5 and 10.0, respectively, and an optimum temperature of 80°C. The Km values for FIVa' and FIVb' were 3.85 and 5.49 mM and the V max values were 2.78 and 2.09 U/mg, respectively, using triacetin as a substrate. The purified lipase fractions retained more than 90% of their activity when stored at room temperature for 36 h. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl 2, FeCl3 and Ellman's reagent. However, 10 mM of CaCl2 and EDTA activated the two purified lipase fractions by 20 to 50%. Both fractions exhibited high specificity towards short- and long-chain fatty acid esters of triacylglycerols. Fraction FIVa' showed higher specificity towards triacetin, tristearin and tripalmitin, whereas fraction FIVb' exhibited higher activity with triacetin, trimyristin and triolein. In addition, the two purified lipase fractions were able to catalyze, to almost the same extent, the hydrolysis of butter and olive, canola and fish oils. The gas-liquid chromatography analysis of free fatty acids, obtained by the hydrolysis of the four edible oils, revealed that fraction FIVa' was more specific for the hydrolysis of fatty acid esters chain lengths of C12 to C18 whereas fraction FIVb' showed a non-specific hydrolyzin

Pseudomonas chlororaphis PA23 is a promising biological control candidate against Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot in canola. A library of transposon mutants was previously created to understand the molecular mechanisms underlying the antifungal capabilities of PA23. A novel LysR-type transcriptional regulator, called PtrA, was identified as a key global regulator involved in secondary metabolite production. The function of PtrA at the molecular level was investigated in this thesis. Solubility problems encountered during the purification of PtrA redirected efforts to studying a truncated version of the protein instead. A two-step purification of the truncated protein, involving streptomycin sulfate precipitation and immobilized metal-ion affinity chromatography, yielded a highly pure protein. Preliminary crystal growth was achieved for the effector binding domain portion of PtrA. Transcriptional fusions suggested that essential regulatory binding sites of ptrA may lie somewhere between 52 and 198 bp upstream of the translational start site. The research presented in this thesis will help guide future functional studies on PtrA.

Pseudomonas aeruginosa is a ubiquitous environmental microorganism that opportunistically colonizes immune-compromised hosts. P. aeruginosa is capable of establishing complex. matrix-encased biofilms during colonization of both environmental and living host surfaces. Biofilms formed by P. aeruginosa are physiologically very different from free-living P. aeruginosa cells, and exhibit increased resistance to environmental stresses, including antibiotic treatment. While the development and establishment of P aeruginosa biofilms has been extensively studied in vitro, several new behaviours of P. aeruginosa in biofilms have recently been observed that may greatly impact on the spread, recolonization and function of biofilms. These processes include bacteriophage mediated lysis and dispersal of P. aeruginosa biofilms, and the generation of phenotypic and genetic variation among bacterial cells that disperse from the biofilm. In this project, the role of bacteriophage activity and phenotypic variation in the development of P. aeruginosa biofilms has been investigated. Induction of a Pf1-like prophage of P aeruginosa (here named Pf4), during biofilm formation was characterized and was shown to increase over the progression of biofilm development. It was observed in this study that the activity of Pf4 caused the emergence of small colony variant (SCY) phenotypes in the effluent run-off from P. aeruginosa biofilms. Computational analysis of the genome ofPf4 resulted in the identification of a novel Toxin-Antitoxin (TA) gene pair, not previously identified within the genome of P. aeruginosa, of which the putative toxin gene product was determined here to play a role in growth-inhibition and the small colony phenotype of P. aeruginosa. TA gene pairs are proposed to induce stress responses in host cells and therefore play a role in survival during periods of environmental stresses such as oxidative or starvation stress. To study the effects of the Pf4 toxin and its possible role in the stress response of P aeruginosa, the Pf4 toxin gene was cloned and placed under the regulation of an inducible arabinose promoter. The proteome expression and biofilm formation as a result of toxin over expression were compared. The proteomic studies performed here indicated that P. acrllginosu biofilms do respond to expression of the toxin component of this putative TA element by increased expression of stress related proteins. Many stress-related groups of proteins were found to be over expressed during induction of the toxin indicating a possible role in stress survival of P. acrllginosa. Homology studies of the Pf4 toxin indicated a strong structural sequence relationship with the toxin ParE of the ParDE TA system. The mode of action of ParE toxin had previously been determined and showed the ParE toxin to be a strong gyrase inhibitor. The Pf4 antitoxin was, however, found to have homology to the Phd antitoxin of the Phd-Doe system of bacteriophage PI. The mode of action of the Doc protein remains to be clearly determined. To better understand the interaction between the Pf4 antitoxin and its cognate toxin protein interaction studies were performed. Peptide fragments of the Pf4 antitoxin were generated for an SPR binding assay and this study identified putative peptide sequences that are responsible for binding of the Pf4 antitoxin to its cognate toxin. Further investigation of a selected strong binding peptide showed that there were 3 key amino acids that were important in binding to the Pf4 toxin, namely His65, Ser67 and A 69 sp. Overall, this study has identified a key role for bacteriophage Pf4 in biofilm development and phenotypic variation in P. aeruginosu, and has provided initial insight into the molecular mechanisms by which this bacteriophage influences growth and gene expression in this organism.

Thesis (M.Eng.). / Written for the Dept. of Civil Engineering and Applied Mechanics. Title from title page of PDF (viewed 2008/05/13). Includes bibliographical references.

Univ., Diss.--Stuttgart, 1992.