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The Production of Species-Specific Highly Unsaturated Fatty Acyl-Containing LCOs from Rhizobium leguminosarum bv. trifolii Is Stringently Regulated by nodD and Involves the nodRL Genes

¹Institute of Biology Leiden, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; ²Department of Mass Spectrometry, Bijvoet Centre for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; ³Australian Research Council Centre of Excellence for integrative Legume Research, GI Group, Research School of Biological Sciences, Australian National University, Sullivan's Creek Road, Acton, ACT 2601, Australia

A proportion of the Nod factors of some Rhizobium leguminosarum bv. trifolii strains is characterized by the presence of highly unsaturated fatty acyl chains containing trans double bonds in conjugation with the carbonyl group of the glycan oligosaccharide backbone. These fatty acyl chains are C_18:3, C_20:3, C_18:4, or C_20:4 and have UV-absorption maxima at 303 and 330 nm. These Nod factors are presumed to be important for host-specific nodulation on clover species. However, in wild-type R. leguminosarum bv. trifolii ANU843, Nod factors with these characteristic acyl chains were not observed using standard growth conditions. They were observed only when nod genes were present in multiple copies or when transcription was artificially increased to higher levels by introduction of extra copies of the transcriptional regulator gene nodD. In a screen for the genetic requirements for production of the Nod factors with these characteristic structures, it was found that the region downstream of nodF and nodE is essential for the presence of highly unsaturated fatty acyl moieties. Mu-lacZ insertion in this region produced a mutant that did not produce detectable levels of the highly unsaturated fatty acyl-bearing Nod factors. The Mu-lacZ insertion was translationally fused to a putative new gene, designated nodR, in the nodE-nodL intergenic region; however, no predicted function for the putative NodR protein has been obtained from data-base homology searches. In a set of 12 wild-type strains of R. leguminosarum bv. trifolii originating from various geographical regions that were analyzed for the presence of a nodR-like gene, it was found that seven strains carry a homologous NodR open reading frame. Taken together, our results suggest a tightly controlled regulation of nod genes, in which we propose that it is the balance of transcriptional levels of nodFE and the nodRL genes that is critical for determining the presence of highly unsaturated fatty acyl moieties in the Nod factors produced by R. leguminosarum bv. trifolii.