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Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3′-phosphoadenosine-5′-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the “household” CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broad-host range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglu-cosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Rhizobium sp. BR816 contains four nodD alleles of which nodD3 is the most important transcriptional regulator for nodulation of Phaseolus vulgaris. Upstream of nodD3 an open reading frame, orf816, was identified. The deduced ORF816 protein shows homology with transcriptional regulators of the AraC/XylS family. The DNA binding domain and the consensus motif, characteristic of the C-terminal region of the members of this family of transcriptional regulators, are present in the deduced ORF816 protein. Activation of nodA gene expression and nodulation of P. vulgaris by Rhizobium sp. NGR234nodD1::omega (Nod-) complemented with the Rhizobium sp. BR816 nodD3 gene were significantly increased in the presence of orf816. This increased nodulation and nod gene induction are mediated through positive regulation of nodD3 expression by ORF816. Expression of orf816 itself is partially RpoN dependent. The role of this transcriptional regulator in the complex cascade regulation of the Rhizobium sp. nodD3 gene is discussed.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.

The broad-host-range, heat-tolerant Rhizobium strain BR816 produces sulphated Nod metabolites. Two ORFs highly homologous to the Sinorhizobium meliloti nodPQ genes were isolated and sequenced. It was found that Rhizobium sp. BR816 contained two copies of these genes; one copy was localized on the symbiotic plasmid, the other on the megaplasmid. Both nodP genes were interrupted by insertion of antibiotic resistance cassettes, thus constructing a double nodP1P2 mutant strain. However, no detectable differences in Nod factor TILC profile from this mutant were observed as compared to the wild-type strain. Additionally, plant inoculation experiments did not reveal differences between the mutant strain and the wild-type. It is proposed that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis suggested that this locus contains genes necessary for the sulphation of amino acids.