Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed March 9, 2008) Graduate School of Arts and Sciences, Dept. of Biology. Includes bibliographical references (p. 60-63).

Siderophores are secreted by marine bacteria to increase Fe uptake when Fe is limiting but are not produced when sufficient Fe is present to saturate growth. These results are well established in laboratory batch cultures of a number of isolates obtained from the open sea. Little is known, however, regarding the kinetics of siderophore secretion by heterotrophic bacteria in response to transients in Fe deprivation and resupply. We examined growth, hydroxamate siderophore concentration, and electron transport chain activity (a biochemical measure of Fe nutritional state) of Pseudoalteromonas haloplanktis, a representative gamma-proteobacterium from the Fe deficient region of the subarctic Pacific Ocean. Hydroxamate concentration was roughly 5-fold higher in batch cultures grown in low than in high Fe medium. Iron injection to the low Fe cultures repressed hydroxamic acid production and increased growth and ETC activity. Steady-state hydroxamate concentration in the chemostat increased 5-fold as Fe-limited growth rate declined from 9.8 to 2.8 d -1. This increase compounded to a 2.8-fold change in hydroxamates cell-1 reflecting the greater costs of growth at low Fe. Three types of Fe perturbation were made to Fe-limited chemostat cultures: 1) A switch perturbation that decreased the dilution rate of the chemostat-by ∼3-fold caused a transient increase in cell density that subsequently declined to a new steady state level. Hydroxamate concentration increased linearly over the same time. 2) A transient addition of dissolved Fe increased the total hydroxamate concentration in the chemostat within 1-3 hours which was followed by a decrease and then subsequent increase as the cells re-entered Fe-limitation. Dilution rate affected the response. Normalized to bacteria density, hydroxamate concentration remained constant for the first 2 hours after the Fe addition and then declined and returned to pre-infusion levels. Thus, Fe addition stimulated siderophore production by increasing the density of bacteria, which continued to secrete hydroxamates at a Fe-limited rate. 3) A continuous addition of low levels of dissolved Fe increased bacteria density and siderophore concentration. The net secretion rate of siderophores was proportional to the increase in Fe supply rate to the chemostat. At high Fe concentration, hydroxamate concentration declined to undetectable levels as the bacteria became Fe-sufficient and C-limited. Siderophore secretion by Fe-limited P. haloplanktis was repressed after 2 hours (corresponding roughly to 1-2 cell generations) following Fe re-supply.

Four strains of the fluorescent pseudomonads were studied to determine the effect of controlled atmospheres on the growth and fluorescent siderophore production at pH 6.0 and 7.8. Bacterial strains were grown in Liquid King's Medium B for 48 hr in the presence of O2/CO2 combination percentages of 21.0/0.03, 18.3/3.0, 15.0/6.0, 12.0/9.0, and 9.0/12.0. The bacterial biomass was determined after centrifugation and the siderophores were isolated, partially purified, and quantified spectrophotometrically. Results showed a steady decline in growth and in siderophore production per unit biomass with decreases in the O2/CO2 ratio at pH 7.8 and to a lesser extent at pH 6.0. The average percentage changes in siderophore production levels, relative to control were +0.8, -1.2, -18.2, and -40.6 at pH 6.0; -33.0, -50.4, -66.8, and -64.1 at pH 7.8 in the presence of O2CO2 levels of 18.0/3.0, 15.0/6.0, 12.0/9.0, and 9.0/12.0, respectively.

No description available.

Thesis (Master of Science)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution,2008. / Bibliography: p. 54-59.

Thesis (Ph. D.)--Washington State University, August 2007. / Includes bibliographical references.

No description available.

N-hydroxylating monooxygenases (NMOs) are Class B flavin-dependent monooxygenases found only in fungi and bacteria. These enzymes catalyze the hydroxylation of nucleophilic primary amines, such as those found in histamine, L-ornithine, L-lysine, and small aliphatic diamines. The hydroxamate moiety produced by this reaction is key for the production of siderophores, small chelating compounds that allow survival in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. Herein we present the characterization of TheA, an NMO from Thermocrispum agreste. The enzyme mechanism was studied using steady state kinetic measurements, thermostability, and stopped flow spectrophotometry assays. Using these techniques, the catalytic rates, substrate binding affinities, thermal stability, and coenzyme specificities were determined. Additionally, NADPH analogues were produced to use as tools to study FAD reduction in NMOs. An unspecific reduction reaction of NADP+ using NaB2H4 yielded [6-2H]-NADPH, [2-2H]-NADPH, and [4-2H]-NADPH. Compound identity was confirmed by mass spectrometry and unidimensional proton nuclear magnetic resonance (NMR). Results presented in this thesis lay the foundation for future studies of NMOs using NADPH analogues. In conjunction, these results will improve the general knowledge and understanding of flavoenzymes, ornithine monooxygenases, and their associated mechanisms. / Master of Science in Life Sciences / Due to the surge of more potent and prevalent microbial pathogens, there is a constant search for new and more specific drugs. One approach is to identify and inhibit enzymes that are key for growth of these pathogens. An example of such enzymes is a group called N-hydroxylating monooxygenases (NMOs) that are key for the production of siderophores, small chelating compounds that allow survival of some fungi and bacteria in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. To develop specific inhibitors of NMOs that can be used as drugs to treat these infections, we first need to understand how these enzymes work. Herein, we characterized a novel NMO from Thermocrispum agrestre. Our results highlight the similarities and differences from previously characterized NMOs. Furthermore, we produced analogues of NADPH, a molecule needed for the mechanism of NMOs. The produced compounds will be used in future studies to understand the step-bystep mechanism of the enzyme. In conjunction, these results will improve the general knowledge and understanding of NMOs and their associated mechanisms and lay the foundation for future studies on the identification of drugs that can be used to treat these diseases.

Graduation date: 1993

Graduation date: 1993