Chemostat analysis was used to investigate induction of the fixABCX genes, grown under limiting oxygen. qRT-PCR revealed that while fixN, a known oxygen responsive gene, is up-regulated fixA is not. Thus low oxygen is not the sole activator of fix gene expression.

Understanding the role of FixABCX in nitrogen

fixation in the Rhizobium leguminosarum symbiosis

Isabel Webb, Rob Green, Elaine Barclay1, Nick Watmough2, Philip Poole3 1. Department of Molecular Microbiology, John Innes Centre, Norwich; 2. University of East Anglia, Norwich; 3. Department of Plant Science, Oxford

Background

• R. leguminosarum bv. viciae 3841 induces nodules on peas (Pisum sativum). Inside these nodules the bacteria differentiate into ‘bacteroids’ and carry out nitrogen fixation. Bacteroids require low oxygen concentrations for nitrogenase to catalyse nitrogen fixation.. Nitrogenase catalyses the following reaction:.

N2 + 8e- + 8H+ + 16ATP 2NH3 + H2 + 16ADP + 16Pi

• The rhizobial fixABCX genes are essential for nitrogen fixation in rhizobia. Their role is not characterised, although their products have homology with mammalian electron transfer proteins1 and electron transfer proteins found in free-living nitrogen fixing species2.

• fixABCX exist as an operon within R. leguminosarum. The genes are thought to be regulated by NifA, a general transcriptional activator of nitrogen fixation • My aim is to understand the role of the FixAB proteins within rhizobial symbioses and understand their transcriptional regulation.

2μm

Mutating the fixABCX operon abolishes nitrogen fixation In frame fixAB mutants strain have been made to ensure the fixCX genes are expressed. Additionally, a fixAB deletion mutant strain was made, using a spectinomycinR cassette insertion, which effectively knocks out the entire fixABCX operon.

References: 1. Watmough, N. J., and Frerman, F. E. (2010) The electron transfer flavoprotein: ubiquinone oxidoreductases. Biochimica et biophysica acta 1797, 1910-1916 2. Edgren, T., and Nordlund, S. (2004) The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to

nitrogenase. J. Bacteriol. 186, 2052-2060

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Future Work

• Step and FLAG tagged FixAB proteins are purified from bacteroids to study their electron transfer characteristics and interacting partners. • The electron dense granules within fixAB bacteroids, suspected to be either lipids or polyphosphate granules, are being investigated. • Additional signals that regulate fixAB expression are being investigated under microaerobic growth.

fixA fixB

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The fixABCX genes are found as a single transcriptional unit in R. leguminosarum

fixABCX mutants show different bacteroid morphology

Nodule phenotype and light and electron micrographs of fix mutants at 28 days post-inoculation. fixABΩspec bacteroids are swollen and non-uniform. ΔfixAB bacteroids are extremely small although uniform. ΔfixAB bacteroids form small, electron-dense granules whose nature is being investigated.

ΔfixAB Ωspec

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Low oxygen does not activate fixABCX gene expression

A model for the role of FixABCX

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The fixABCX promoter region coupled to LuBCDABE, allows screening for Fix gene expression under varying conditions. Luminescence can be seen in nodules at 4 weeks post inoculation. Luminescence is not seen in free-living cultures.

This work has been carried out as part of a BBSRC-funded Doctoral Training Grant. Thanks also go to the Biochemical Society for providing a travel grant to present this work.

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Using the lux system as a gene expression indicator