UBC Theses and Dissertations
Amino acid transport and pool formation in Pseudomonas aeruginosa Kay, William Wayne
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.
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