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Functional and Regulatory Analysis of the Two Copies of the fixNOQP Operon of Rhizobium leguminosarum Strain VF39

¹Ökologie des Bodens, Botanisches Institut, RWTH Aachen, Worringerweg 1, D-52056 Aachen, Germany; ²Department of Biological Sciences, University of Calgary, Calgary AB, T2N 1N4, Canada; and ³Agriculture Canada, Lethbridge Research Station, BOX 2000 Main, Lethbridge AB, T1J 4B1, Canada

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the hemecopper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixNgusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation.

Additional keywords: Fnr-like regulators.