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Pathways of aromatic metabolism in Rhizobium

Chen, Yung-Pin (1986) Pathways of aromatic metabolism in Rhizobium. PhD thesis, Murdoch University.

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Studies on the oxidation of a variety of aromatic substrates by Rhizobium trifolii TA1 and Rhizobium leguminosarum MNF3841 indicated that the former could oxidise aromatic substrates normally leading to the catechol or protocatechuate branches of the 3-oxoadipate pathway, while the latter could not use substrates like benzoate requiring the catechol branch. Assays for typical enzymes of the catechol and protocatechuate branches confirmed this conclusion. The detection of a catechol 2,3-dioxygenase only in R. trifolii TA1 further suggested the existence of a meta-cleavage pathway, though no further characterization of such a pathway was attempted.

The uptake systems for benzoate and 4-hydroxybenzoate were studied specifically in R. trifolii TA1 and for 4-hydroxybenzoate in R. leguminosarum. Both systems are active in nature, and both have a pattern of inhibition by aromatic substrates. The two transport systems were not cross-inducible in R. trifolii, but were induced by growth on the appropriate substrate. The transport systems for 4-hydroxybenzoate and protocatechuate also appeared to be distinct. A mutant defective in 4-hydroxybenzoate transport was also isolated.

Removal of periplasmic proteins by lysozyme-EDTA treatment of R. trifolii TA1 abolished 4-hydroxy-benzoate uptake activity in cells grown on 4-hydroxy-benzoate. Cells grown on succinate and treated similarly continued to transport succinate. The periplasmic protein fraction from 4-hydroxybenzoate-grown cells bound radioactive 4-hydroxybenzoate, but similar fractions from cells grown on glucose, succinate, protocatechuate or benzoate did not. A protein was purified from the hydroxybenzoate-grown cells which bound up to 1 mole of [l4C}-4-hydroxy-benzoate per mole of protein. It has been suggested that a binding protein may be required for the transport of 4-hydroxybenzoate.

The two dioxygenases involved in cleavage of the aromatic ring - protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase were purified from R. trifolii TA1, and some of their biochemical properties established. In general, they show considerable resemblance to comparable enzymes purified from Pseudomonas species. Both appeared to be dependent on Fe(III) for activity; the iron could be removed from them by 1,10-phenanthroline after reduction, and activity restored only with an EDTA chelate of Fe(III). Both were extremely sensitive to inhibition by the EDTA chelates of Ni(III) and Al(III) where EDTA itself was ineffective. It has been suggested that inhibition of the dioxygenases by soil aluminium in highly acid soils might well prevent utilization of aromatic compounds by rhizobia and lead to poor survival.

The regulation of the pathways from 4-hydroxy-mandelate and L-mandelate to 4-hydroxybenzoate and catechol, respectively, was studied. Unlike the situation in many Pseudomonas species, the pathways from mandelate and 4-hydroxymandelate appear to involve quite distinct enzymes in R. trifolii TA1.

Studies of enzyme induction in the 4-hydroxy-mandelate and mandelate pathways to protocatechuate and catechol, respectively, suggested that induction of enzymes was sequential for all enzymes, though only a limited range of substrates was available for growth.

When cells were grown on particular substrates, and another substrate downstream from it added, the enzymes upstream showed lowered activities. This finding was true for both the mandelate and hydroxymandelate pathways and for those segments of the 3-oxoadipate pathway which were tested.

Cells grown on aromatic substrates in the presence of other carbon sources showed lowered activities of the enzymes of aromatic degradation. These activities occurred whether the non-aromatic substrate lower were a product of aromatic degradation (succinate or acetate) or glucose. It was not clear whether this represented an end-product type of repression or a limited catabolite repression.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Environmental and Life 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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