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Genetic and physiological studies on the entry of aromatic substrates into cells of Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii

Wong, Cheryl (1993) Genetic and physiological studies on the entry of aromatic substrates into cells of Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii. PhD thesis, Murdoch University.

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The entry of aromatic substrates into Rhizobium leguminosarum biovar viciae MNF300 and Rhizobium leguminosarum biovar trifolii WU95 has been investigated. Two independently-obtained Tn5- insertion mutants of R. leguminosarum bv viciae, MNF3030 and MNF3036, and one Tn5-233-insertion mutant of R. leguminosarum bv trifolii, MNF9013, each unable to grow on 4-hydroxybenzoate as sole carbon source, were used to study the transport of 4- hydroxybenzoate and protocatechuate. All of these mutants were unable to take up labelled 4-hydroxybenzoate but were able to transport labelled protocatechuate.

Enzyme assays on the mutants revealed that they lacked 4-hydroxybenzoate hydroxylase (PobA) activity, suggesting that uptake could be via metabolic drag. However, inhibition of 4-hydroxybenzoate uptake in the wild-type using metabolic poisons and competitive inhibitors suggested that uptake was via an active system. To determine how 4-hydroxybenzoate entered these cells, the mutated gene was isolated by complementing the mutants with a gene library of R. leguminosarum by viciae B155. Complementation simultaneously restored both the ability of the mutants to take up and grow on 4-hydroxybenzoate as sole carbon source and the activity of 4-hydroxybenzoate hydroxylase. In addition, the region of DNA flanking Tn5 in one of the mutants was cloned.

Hybridization studies revealed that a labelled 4.0 kb DNA fragment isolated from the B155 gene library and able to complement the mutants hybridized to two DNA fragments (2.2 kb and 1.8 kb) of an EcoR1 genomic digest of MNF300 DNA and to two DNA fragments (2.2 kb and 7.6 kb) of EcoR1 genomic digests of the mutants MNF3030 and MNF3036. This pattern indicated that an EcoR1 site present in MNF300 was absent from B155 and that Tn5 (5.8 kb) had inserted into the 1.8 kb fragment in MNF3030 to produce the 7.6 kb band. Using labelled fragments of the 4.0 kb B155 complementing DNA, the 4.0 kb fragment was mapped. A 2.0 kb EcoR1/Pst1 fragment, which encompassed the 1.8 kb fragment in MNF300 into which Tn5 had inserted in the mutants, also had the capacity to complement the mutants MNF3030, MNF3036 and MNF9013, restoring their ability to grown on 4-hydroxybenzoate. The 2.0 kb fragment of B155 and 1.5 kb of DNA sequences flanking Tn5 in MNF3030 were sequenced.

The 2.0 kb B155 complementing DNA contained only one complete gene; it showed a high degree of homology to sequences for pobA (4-hydroxybenzoate hydroxylase) found in the database GenBank for Pseudomonas aeruginosa and Acinetobacter calcoaceticus. DNA sequences from the mutant MNF3030 showed that approximately 80% of the pobA gene had been cloned and that Tn5 had inserted directly into the gene. In addition, the amino acid sequences derived from DNA sequences upstream of pobA on the complementary strand revealed homology to pobR, the regulatory gene for pobA in Acinetobacter calcoaceticus.

The evidence supporting an active uptake system was found to be explicable in terms of direct effects on 4-hydroxybenzoate hydroxylase, since both hydroxyaromatic analogues of 4-hydroxybenzoate and metabolic poisons also inhibited the enzyme. It is therefore proposed that 4-hydroxybenzoate enters cells of R. leguminosarum by diffusion, either passively or facilitated by a binding protein. Induction of the pathway for 4-hydroxybenzoate catabolism occurs in response to 4-hydroxybenzoate, possibly by the binding of this substrate to the product of pobR, which induces a conformational change enabling the transcription of pobA. The product of pobA, 4-hydroxybenzoate hydroxylase, converts 4-hydroxybenzoate to protocatechuate and the further metabolism of this substrate allows the entry of more 4-hydroxybenzoate. Thus 4-hydroxybenzoate is "pulled" into the cell by metabolic "drag".

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Dilworth, Michael and Glenn, Andrew
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