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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL TECHNOLOGIES FOR PRODUCING RENEWABLE CHEMICALS AND POLYMERS FROM CARBOHYDRATES DERIVED FROM AGRICULTURAL FEEDSTOCKS

Location: Renewable Product Technology Research Unit

Title: CBS domain-containing proteins are Rhizopus oryzae ferrioxamine receptors

Authors
item Liu, M -
item Gebremariam, T -
item Skory, Christopher
item Edwards, Jr, J -
item Ibrahim, A -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 12, 2012
Publication Date: September 12, 2012
Citation: Liu, M., Gebremariam, T., Skory, C.D., Edwards, Jr, J., Ibrahim, A. 2012. CBS domain-containing proteins are Rhizopus oryzae ferrioxamine receptors [abstract]. Interscience Conference on Antimicrobial Agents and Chemotherapy. M-973. p. 108.

Technical Abstract: Background: Iron-overload patients treated with deferoxamine are uniquely susceptible to mucormycosis, because Rhizopus spp. can obtain iron from ferrioxamine (deferoxamine + Fe**3+). Previously we have identified two closely related, ferrioxamine-inducible R. oryzae genes (FOB1 and FOB2) in which their proteins bound ferrioxamine in vitro. We sought to determine if these two genes encode the ferrioxamine receptor(s). Methods: RNAi was used to silence FOB1 or FOB2 expression individually or collectively. For individual silencing, two plasmids were generated in plasmid pRNAi-pdc by cloning a 450-bp fragment unique to each gene and its corresponding inverted repeat separated by a 100-bp intron (total of 1 kb). These two constructs are termed pFOB1 and pFOB2, respectively. To silence both genes at the same time, the 1 kb sequence from pFOB1 was cloned downstream of pFOB2, separated by a 50-bp interval to yield pFOB1/2. Individual constructs were transformed into R. oryzae pyrF mutant and transformants were selected on medium lacking uracil. Transformants were characterized for ferrioxamine gene expression, growth on medium with ferrioxamine as a sole source of iron, and **55Fe uptake from ferrioxamine. Results: qRT-PCR showed that R. oryzae transformed with either pFOB1 or pFOB2 had 80-85% inhibition in the corresponding gene without affect the expression of the other gene. These transformants had reduced growth between 20-25% on medium supplemented with 10 micromolar ferrioxamine and 45-75% inhibition in **55Fe uptake compared to control cells transformed with empty plasmid. In contrast, cells transformed with the dual construct (pFOB1/2), had up to 95% inhibition in expression of both FOB1 and FOB2. Dual inhibition transformants had 45% inhibition in growth on medium with ferrioxamine and up to 85% reduction in **55Fe uptake compared to control cells. Conclusion: Our results suggest that these two CBS domain-containing proteins represent the ferrioxamine receptors. Further studies to determine the effect of FOB1 and FOB2 silencing on R. oryzae virulence in deferoxamine-treated mice are underway.

Last Modified: 4/19/2014
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