Inositol catabolism, a key pathway in sinorhizobium meliloti for competitive host nodulation

Abstract

The nitrogen-fixing symbiont of alfalfa, Sinorhizobium meliloti, is able to use myo-inositol as the sole carbon source. Putative inositol catabolism genes (iolA and iolRCDEB) have been identified in the S. meliloti genome based on their similarities with the Bacillus subtilis iol genes. In this study, functional mutational analysis revealed that the iolA and iolCDEB genes are required for growth not only with the myo-isomer but also for growth with scyllo- and d-chiro-inositol as the sole carbon source. An additional, hypothetical dehydrogenase of the IdhA/MocA/GFO family encoded by the smc01163 gene was found to be essential for growth with scyllo-inositol, whereas the idhA-encoded myo-inositol dehydrogenase was responsible for the oxidation of d-chiro-inositol. The putative regulatory iolR gene, located upstream of iolCDEB, encodes a repressor of the iol genes, negatively regulating the activity of the myo- and the scyllo-inositol dehydrogenases. Mutants with insertions in the iolA, smc01163, and individual iolRCDE genes could not compete against the wild type in a nodule occupancy assay on alfalfa plants. Thus, a functional inositol catabolic pathway and its proper regulation are important nutritional or signaling factors in the S. meliloti-alfalfa symbiosis.

FIG. 1.

The proposed myo-inositol catabolic pathway (http://www.genome.jp/kegg/). Compound 1, myo-inositol (MI); compound 2, 2KMI; compound 3, 3D-(3,4/5) trihydroxycyclohexane-1,2-dione (THcHDO); compound 4, 5-deoxy glucuronic acid (5DG); compound 5, 2-deoxy-5-keto-d-gluconic acid (DKG); compound 6, DKGP; compound 7, dihydroxyacetone phosphate (DHAP); compound 8, malonic semialdehyde (MSA); compound 9, acetyl coenzyme A (acetyl-CoA). Enzymes: IdhA, myo-inositol dehydrogenase; IolE, 2KMI dehydratase; IolD, THcHDO hydrolase; IolB, 5DG isomerase; IolC, DKG kinase; IolJ aldolase (not yet identified in S. meliloti); IolA, MSA dehydrogenase.

FIG. 2.

Ability of S. meliloti wild-type and the idhA, smc01163 (1163), iolR, iolC, iolD, iolE, iolA, and iolB mutant strains to grow with 0.2% myo-inositol, d-chiro-inositol, scyllo-inositol, or 2KMI as the sole carbon source in minimal medium. Open reading frames are depicted as open arrows. The locations of the mini-Tn5 insertions in the S. meliloti 2011 mutants are marked by vertical arrowheads. The star indicates the position of the plasmid insertion in the S. meliloti iolB mutant. Horizontal arrows above the genes indicate predicted transcriptional units. Each mutant's ability (+) or inability (−) to use inositol compounds as the sole carbon source is indicated.

FIG. 3.

β-Glucuronidase activities of the S. meliloti idhA (A), smc01163 (B), and iolR (C) gusA reporter gene fusions in the respective mutant strains. The reaction rate is expressed in nmol p-nitrophenol produced per minute per OD₆₀₀ unit. Cultures were grown in minimal medium containing 0.2% of the following carbon sources: myo-inositol (MI), 2KMI, glycerol (Gly), glucose (Glu), succinate (Suc), or combinations thereof. Bars represent the averages of two independent experiments, and error bars denote SEM. MI* indicates that the idhA mutant did not grow with myo-inositol as the sole carbon source in minimal medium, but the residual β-glucuronidase activity is probably due to the carryover of cells from the TY preculture.

FIG. 4.

Competition assay for nodule occupancy. The S. meliloti 2011 idhA (WIDHA), smc01163 (W63-1 and W63-2), iolR (WIOLR), iolC (WIOLC), iolD (WIOLD), iolE (WIOLE), iolA (WIOLA), and glyA2 (WGLYA) mutant strains were inoculated on alfalfa plants in a 1:1 ratio with the wild type. After 20 weeks nodules were harvested and surface sterilized, and rhizobia were reisolated from the nodules. The wild-type versus the mutant output ratio of the reisolated rhizobia was determined via selective plating. Bars represent the averages of two independent experiments representing nodules from six plants each. Error bars denote SEM.