The RpiR-like repressor IolR regulates inositol catabolism in Sinorhizobium meliloti

Abstract

Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa, has the ability to catabolize myo-, scyllo-, and D-chiro-inositol. Functional inositol catabolism (iol) genes are required for growth on these inositol isomers, and they play a role during plant-bacterium interactions. The inositol catabolism genes comprise the chromosomally encoded iolA (mmsA) and the iolY(smc01163)RCDEB genes, as well as the idhA gene located on the pSymB plasmid. Reverse transcriptase assays showed that the iolYRCDEB genes are transcribed as one operon. The iol genes were weakly expressed without induction, but their expression was strongly induced by myo-inositol. The putative transcriptional regulator of the iol genes, IolR, belongs to the RpiR-like repressor family. Electrophoretic mobility shift assays demonstrated that IolR recognized a conserved palindromic sequence (5'-GGAA-N6-TTCC-3') in the upstream regions of the idhA, iolY, iolR, and iolC genes. Complementation assays found IolR to be required for the repression of its own gene and for the downregulation of the idhA-encoded myo-inositol dehydrogenase activity in the presence and absence of inositol. Further expression studies indicated that the late pathway intermediate 2-keto-5-deoxy-D-gluconic acid 6-phosphate (KDGP) functions as the true inducer of the iol genes. The iolA (mmsA) gene encoding methylmalonate semialdehyde dehydrogenase was not regulated by IolR. The S. meliloti iolA (mmsA) gene product seems to be involved in more than only the inositol catabolic pathway, since it was also found to be essential for valine catabolism, supporting its more recent annotation as mmsA.

Fig. 1.

Suggested inositol catabolic pathway in S. meliloti. Compounds: [I], d-chiro-inositol; [II], myo-inositol; [III], scyllo-inositol; [IV], 2-keto-myo-inositol; [V], 3-d-(3,4/5)-trihydroxycyclohexane-1,2-dione; [VI], 5-deoxy glucuronic acid; [VII] 2-deoxy-5-keto-d-gluconic acid; [VIII] 2-deoxy-5-keto-d-gluconic acid 6-phosphate; [IX] dihydroxyacetone phosphate; [X], malonate semialdehyde (MSA); [XI], acetyl coenzyme A. Enzymes: IdhA, myo-inositol dehydrogenase; IolY, scyllo-inositol dehydrogenase; IolE, 2KMI dehydratase; IolD, 3-d-(3,5/4)-trihydroxycyclohexane-1,2-dione hydrolase; IolB, 5-deoxy-glucuronate isomerase; IolC, 2-deoxy-5-keto-d-gluconic acid kinase; IolA (MmsA), methylmalonate semialdehyde dehydrogenase.

Fig. 2.

IolR-DNA-binding assay. Increasing concentrations of purified IolR-His₆ protein (indicated at the top of each lane) were incubated with 10 ng of 8 different PCR products. The positions of free DNA (open arrowheads) and IolR-DNA complexes (solid arrowheads) are indicated. As a control, 0.3 μM protein without DNA in binding buffer was loaded. (A) EMSA with the upstream regions of idhA, iolY, iolR, iolC, iolA, and nodD1 (IolR-independent control). (B) The identified IolR-binding motifs of the idhA, iolY, iolR, and iolC genes; controls 1 and 2, DNA fragments containing the sequences up- and downstream of the IolR-binding motif of iolY, respectively; lanes M, 100-bp ladder.

Fig. 3.

Transcriptional organization of S. meliloti inositol catabolic genes. The 2011 wild-type strain was grown in minimal medium with myo-inositol (MI) or glycerol (Gly) as the sole carbon source. The total RNA was purified and used as a template in RT-PCR (RT). cDNA was amplified with primers flanking the indicated intergenic regions of the iolYRCDEB cluster and the intragenic regions of the idhA, iolA, and smc01981 (cytochrome c [cC]) genes. Boxes represent the individual inositol genes, and arrows represent transcriptional units. As controls, PCR was performed with RNA samples (no RT) and no template (H₂O).

Fig. 4.

β-Glucuronidase activities of the S. meliloti iolR-, iolD-, and iolE-gusA reporter gene fusions in the respective mutant strains. The WIOLR/pTE3, WIOLD/pTE3, and WIOLE/pTE3 strains harbor the empty vector pTE3 as a control, while the WIOLR/pIOLR, WIOLD/pIOLD, and WIOLE/pIOLE strains express the corresponding wild-type genes from pTE3. The reaction rate is expressed in nmol p-nitrophenol produced per minute per OD₆₀₀ unit. Bars represent the average of two independent experiments, and error bars denote ±SEM. Cultures were grown in minimal medium containing glycerol (Gly) or myo-inositol (MI) as the sole carbon source.

Fig. 5.

NAD(H)-dependent myo-inositol dehydrogenase assay with crude cell extracts obtained from S. meliloti wild-type and mutant strains grown in minimal medium containing 0.2% glycerol (Gly) or myo-inositol (MI) as the sole carbon source. The reaction rate is expressed in nmol NAD⁺ reduced per minute per mg protein. Bars represent the average of two independent experiments, and error bars denote ±SEM. The WIOLC/pTE3, WIOLD/pTE3, WIOLE/pTE3, and TIOLB/pTE3 strains did not grow in minimal medium with myo-inositol as the sole carbon source.

Fig. 6.

β-Glucuronidase activities of the S. meliloti iolA-gusA reporter gene fusion in the iolA mutant strain. The WIOLA mutant was grown in minimal medium containing myo-inositol (MI), glycerol (Gly), glucose (Glu), succinate (Suc), or combinations thereof at a final concentration of 0.2% as the carbon source. Bars represent the average of two independent experiments, and error bars denote ±SEM.

Fig. 7.

Growth of S. meliloti wild-type strain 2011 (open circles) and the corresponding iolA mutant (solid squares) with 0.2% valine as the sole carbon source in minimal medium. The optical density was determined spectrophotometrically at 600 nm. Bars represent the average of two experiments, and error bars denote ±SEM.

Fig. 8.

Model for IolR-mediated iol gene regulation. IolR binds to the conserved binding motif GGAAN₆TTCC in the regulatory region of the idhA gene and in the three regulatory regions of the iolYRCDEB operon in the absence of inositol, but it does not repress transcription completely. Inositol is being catabolized as soon as it is imported into the cell, and the late pathway intermediate, 2-deoxy-5-keto-d-gluconic acid 6-phosphate, antagonizes the IolR-mediated transcriptional repression. The mmsA (iolA) gene is constitutively expressed and is not subject to IolR regulation, but mmsA expression is nevertheless increased in the presence of inositol.