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|Title:||The uptake and catabolism of aromatic compounds by bacteria.|
|Authors:||Jones, David Charles Noel.|
|Presented at:||University of Leicester|
|Abstract:||The microbial catabolism of two aromatic acids, phenylacetate and 3-hydroxybenzoate, has been studied. A variety of classical biochemical and genetic techniques have been used to eliminate several putative pathways for the catabolism of phenylacetate in Escherichia coli K10 and Pseudomonas putida U. A phenylacetate negative mutant of E.coli, previously isolated in this laboratory, was shown to accumulate 2-hydroxyphenylacetate when exposed to phenylacetate. The mutation responsible for the inability of this mutant to grow on phenylacetate has been mapped on the E.coli K10 chromosome to about minute 30.4. To facilitate the study of 3-hydroxybenzoate transport and degradation, a method was developed microsynthesis of radiolabelled [14c] 3-hydroxybenzoate of high specific activity. By a combination of substrate oxidation experiments by whole cells, enzyme assays and tracer experiments with radiolabelled 3-hydroxybenzoate, it has been demonstrated that K. pneumoniae degrades 3-hydroxybenzoate via an inducible pathway in which 2,5-dihydroxybenzoate, maleylpyruvate and fumarylpyruvate are intermediates. Experiments suggest that 2,5-dihydroxybenzoate and/or maleylpyruvate are the inducers of the pathway. The synthesis of [14c-carboxyl] 3-hydroxybenzoate also enabled the study of various aspects of the uptake of 3-hydroxybenzoate by K. pneumoniae. It was shown that the uptake of 3-hydroxybenzoate was mediated by a permease induced during growth on either 3-hydroxybenzoate or 2,5-dihydroxybenzoate. The permease displayed saturation kinetics with a Ks value of approximately 1,75 uM for 3-hydroxybenzoate and could transport 3-hydroxybenzoate against a concentration gradient. Uncouplers inhibited the uptake of 3-hydroxybenzoate demonstrating that the driving force for this active transport system was the proton motive force across the cellular membrane. No evidence was gained which would suggest that a general aromatic acid permease was induced during growth of K. pneumoniae on 3-hydroxybenzoate, but the 3-hydroxybenzoate permease may be capable of transporting some substrates of similar structure to 3-hydroxybenzoate.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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