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Sidero[p]hore synthesis by Rhizobium leguminosarum biovar viciae WSM710

Worsley, Penelope S. (2000) Sidero[p]hore synthesis by Rhizobium leguminosarum biovar viciae WSM710. PhD thesis, Murdoch University.

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PW7007 cells were grown in media containing 20 µM iron, then transferred to media containing no added iron to monitor the induction of siderophore biosynthetic genes. A rise in the expression of the vbsS gene above basal level was measurable 4 h post-transfer in control cultures. Addition of the potential precursor amino acids L-glutamate and Lomithine or the amino acid L-arginine did not alter the time or extent of induction of expression.

Addition of the siderophores vicibactin, desferrioxamine, enterobactin or vicibactin/enterobactin together caused expression of the siderophore synthesis gene to be measurable earlier than in control cultures, at 2 h post-transfer. This early induction is probably due to the siderophores binding all the residual iron in the low-iron cultures and rendering it ‘invisible’ to the cells until suitable outer membrane receptors and uptake proteins can be synthesised. Addition of citrate to the low-iron cultures caused the induction of synthesis genes to occur at the same time as control cultures (4 h post-transfer), but with a reduced level of expression. The citrate-iron complex is constitutively taken up by WSM710 and so can immediately supply some of the cell’s iron needs.

Expression of vbsS was repressed by the addition of iron to cells growing in low-iron medium, regardless of whether cells had iron reserves (cells that had been grown in high iron medium prior to transfer into low iron medium) or no iron reserves (cells which had been grown in low iron medium prior to subculture in low iron medium). It is suggested that this is because synthetase activity is non-rate-limiting for the synthesis of vicibactin; cells can still manufacture vicibactin even with a low residual level of synthetase.

The synthesis of vicibactin was then studied in planta through monitoring the expression of the labelled siderophore biosynthetic gene (vbsS) in PW7007. Vetch plants were inoculated with WSM710 or PW7007 (which differ only in their siderophoreproducing abilities) and grown in low and high iron hydroponic media. Analysis of the nitrogen and iron concentration in plant tops, the weight of plant tops, the number of nodules per plant and the histological characteristics of those nodules in terms of bacteroid content, revealed no inoculant-based difference between plant nodules with bacteroids from a strain able to synthesise siderophores and those nodulated with a siderophore-negative strain. The only inoculant-based difference detected was a higher level of iron accumulated in nodules containing PW7007 bacteroids compared with nodules occupied by WSM710. Even nodules containing PW7007 from plants grown in low-iron medium had more iron than nodules containing WSM710 from plants grown in high iron medium. The reason for this accumulation of iron in nodules occupied by non-siderophore producing cells is currently unexplained.

Nodules harvested from plants grown in high and low iron-media and containing PW7007 were stained to assay activity of the gusA reporter gene (an indicator of vbsS expression). Nodules from both iron-deficient and iron-sufficient plants stained deep blue. However, after the bacteroids were extracted and assayed for specific activity, the level of actual gusA activity was found to be very low, at levels similar to those of uninduced laboratory-grown bacterial cultures. The intense colour development seen in whole nodule stains was probably due to low-level basal expression of (3-glucuronidase leading to product building up within the nodule. This emphasises the need for specific activities to be assayed on extracted bacteroids, so that a true picture of the level of expression of marked genes, such as those involved in siderophore production, can be determined. The lack of expression of the vicibactin biosynthetic gene, vbsS, shows that siderophore synthesis in nodules does not occur even in severely iron-deficient plants.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Division of Science and Engineering
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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