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Iron nutrition of Rhizobium leguminosarum biovar viciae MNF710

Carson, Kerry C.A. (1993) Iron nutrition of Rhizobium leguminosarum biovar viciae MNF710. PhD thesis, Murdoch University.

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This thesis reports investigations into the iron nutrition and metabolism of Rhizobium leguminosarum biovar viciae MNF710; this organism excretes a trihydroxamate siderophore which I have named hydroxamate K This is the first report of a trihydroxamate siderophore produced by root nodule bacteria.

Using transposon Tn5 mutagenesis, three mutants of MNF 710were isolated, two of which proved useful. Mutant MNF7101 excretes a variety of hydroxamate siderophores but not hydroxamate K The major product is a trihydroxamate chemical different from hydroxamate K but still structurally similar.

Hydroxamate K is a trihydroxamic acid, which appears to consist of three molecules each of N5-hydroxyornithine and 3-hydroxybutyrate, joined together by alternating peptide and ester linkages. The N5-hydroxyornithine is also acetylated to produce the hydroxamic acid groups involved in iron chelation. The major trihydroxamate siderophore produced by mutant MNF7101 carries a positive charge and lacks the acetyl groups found in hydroxamate K MNF7101 therefore appears to lack a specific acetylase activity required for the acetylation of N5 - hydroxyornithine in MNF710.

Mutant MNF7102 does not produce hydroxamate K or any other siderophore, but can still transport 55Fe complexed with it. This mutant appears to be defective in either the induction of hydroxamate K biosynthesis, or in a vital biosynthtic gene.

Iron deficiency in MNF710 induces the production of three iron-repressible outer membrane proteins (IROMPs) which are probably receptors for iron- siderophore complexes. Cells of MNF710 can transport 55Fe complexed to hydroxamate K, ferrioxamine B, ferrichrome, ferrichrome A, rhodotorulate, citrate and nitrilotriacetic acid (NTA). MNF7102 will also transport these iron-siderophore complexes. Neither MNF710 nor MNF7102 will transport 55Fe complexes to enterobactin or to the pyoverdines from Pseudomonas aeruginosa ATCC15692 or ATCC17400.

In R. leguminosarum MNF710 all of these transport systems are ironregulated, but in MNF7102 iron transport mediated by rhodotorulate, citrate and NTA is constitutive.

Other root nodule bacteria, like Rhizobium leguminosarum biovar trifolii WU95 and Rhizobium meliloti U45, also transport 55Fe complexed to siderophores they do not produce. In some instances these transport systems are iron-repressible (hydroxamate K, ferrioxamine B and ferrichrome transport in WU95) and in others they are constitutive (rhodotorulate, citrate and NTA transport in both WU95 and U45, and ferrichrome and ferrichrome A transport in U45).

When grown with a high iron concentration, R. leguminosarum MNF710 appears to accumulate iron in the form of a bacterioferritin and other iron storage proteins identifiable by Mossbauer spectroscopy. While this is the first indication of iron storage proteins in root nodule bacteria, the roles these may play in the regulation of iron metabolism within the cells are unknown.

Biosynthesis of hydroxamate K is iron-regulated, with excretion occurring only when cells of R. leguminosarum MNF710 are iron-stressed. However, induction of the biosynthetic genes for hydroxamate K appears to be influenced by some form of stored iron within the cells. A model has been developed in which hydroxamate K production is controlled by an "immediately available" form of iron which is in dynamic equilibrium with the storage iron. Regardless of the external iron concentration it appears that the iron concentration within the cell must be maintained above a critical level; if not, siderophore synthesis is derepressed and continues until the stored iron concentration is restored.

The significant finding from this work is that the free-living form of R. leguminosamm MNF710 has the potential to use three different strategies to ensure survival and growth under iron-deficient conditions - specific siderophore production and uptake, utilization of siderophores produced by other microorganisms, and short-term iron storage for later growth.

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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