An orphan LuxR homolog of Sinorhizobium meliloti affects stress adaptation and competition for nodulation

Abstract

The Sin/ExpR quorum-sensing system of Sinorhizobium meliloti plays an important role in the symbiotic association with its host plant, Medicago sativa. The LuxR-type response regulators of the Sin system include the synthase (SinI)-associated SinR and the orphan regulator ExpR. Interestingly, the S. meliloti Rm1021 genome codes for four additional putative orphan LuxR homologs whose regulatory roles remain to be identified. These response regulators contain the characteristic domains of the LuxR family of proteins, which include an N-terminal autoinducer/response regulatory domain and a C-terminal helix-turn-helix domain. This study elucidates the regulatory role of one of the orphan LuxR-type response regulators, NesR. Through expression and phenotypic analyses, nesR was determined to affect the active methyl cycle of S. meliloti. Moreover, nesR was shown to influence nutritional and stress response activities in S. meliloti. Finally, the nesR mutant was deficient in competing with the wild-type strain for plant nodulation. Taken together, these results suggest that NesR potentially contributes to the adaptability of S. meliloti when it encounters challenges such as high osmolarity, nutrient starvation, and/or competition for nodulation, thus increasing its chances for survival in the stressful rhizosphere.

FIG. 1.

Alignment of the deduced protein sequence of NesR with those of selected LuxR-type proteins. The sequences of Agrobacterium tumefaciens TraR (Entrez protein database accession no. AAA64793; 14% identity), Vibrio fischeri LuxR (AAA27542; 20% identity), Pseudomonas aeruginosa RhlR (AAG06865; 19% identity), Sinorhizobium meliloti ExpR (NP_387385/NP_387388; 17% identity) and SinR (NP_385944; 22% identity), Xanthomonas oryzae pv. oryzae OryR (Q5H3E9; 42% identity), and Xanthomonas campestris pv. campestris XccR (AAY48364; 44% identity) are aligned with that of S. meliloti SMc04032 (NesR). The alignment was performed using Vector NTI Advance 10 (Invitrogen) software. Shaded black letters represent highly similar residues; shaded white letters represent identical residues. Asterisks indicate conserved residues of LuxR-type proteins (80).

FIG. 2.

Active methyl cycle and catabolism of glycine betaine in S. meliloti. Cyclic synthesis of methionine to produce the potent methyl donor SAM is known as the active methyl cycle. In S. meliloti, methionine generated from either methyl tetrahydrofolate or glycine betaine is converted to SAM, which, after sequential demethylations, regenerates homocysteine. The production of methionine from glycine betaine also yields dimethylglycine and is the first step of the catabolic degradation pathway of glycine betaine to pyruvate. The pathway and its associated genes are adapted from information provided by the KEGG database, the S. meliloti Rm1021 genome sequence, Barra et al., and Smith et al. (7, 26, 67). M-THF, methyl tetrahydrofolate; THF, tetrahydrofolate; ^5,10-M-THF, N⁵,N¹⁰-methylene tetrahydrofolate.

FIG. 3.

nesR regulates the expression of genes from the active methyl cycle of S. meliloti. Quantitative real-time PCR assays were performed to measure the expression of genes from the active methyl cycle. Changes in expression were calculated as 2^−ΔΔC^T from the C_T values obtained by real-time PCR analyses. Negative change values indicate downregulation in the nesR mutant (Rm8530 nesR) compared to expression in the wild-type strain (Rm8530). The expression of the downregulated genes was restored when the mutant was complemented with nesR on a plasmid (Rm8530 nesR + pJNesR). Rm8530 nesR + pJN105 served as a vector-only control. Results are averages from at least three independent biological experiments, where within the replicates the coefficient of variance of the C_T values was <4%. The experiments included the SMc00128 gene as an internal control (38).

FIG. 4.

Analysis of sensitivity to stress. The nesR mutant showed reduced efficiency in adapting to an osmotic upshock and detergent stress. (A) Growth curves of the wild-type strain (Rm8530), the nesR mutant, and complemented strains in minimal low-phosphate medium supplemented with 0.5 M NaCl. Results are means for three independent experiments, and calculated standard errors are indicated. (B) To test for detergent stress, the nesR mutant and the wild-type strain were subjected to increasing concentrations of DOC by plating onto LB-DOC agar, and the resulting CFU was determined. Results are means for three independent experiments; standard deviations from the means are shown. The differences between the wild-type and mutant CFU were significant (P < 0.02).

FIG. 5.

Growth with glycine betaine as a sole carbon source. S. meliloti has the unique ability to catabolize glycine betaine as a sole source of carbon and energy. Growth analyses of the wild-type strain, the nesR mutant, and complemented strains inoculated in minimal low-phosphate medium where mannitol was replaced with 1 mM glycine betaine are shown. Results are means for three independent experiments, and calculated standard errors are indicated.

FIG. 6.

Symbiosis and competition for plant nodulation. The nesR mutant was capable of establishing symbiosis but was less proficient at nodule occupancy when competing with the wild-type strain. M. sativa roots were coinoculated with different ratios of the wild-type strain (Rm8530) and the nesR mutant on Jensen's medium (41). Percentages of wild-type and mutant strains recovered from plant nodules at different inoculum ratios were compared. The percentages of bacteria applied to the plants are shown along the x axis, and the percentages recovered from crushed nodules are shown along the y axis. The differences observed were significant (P < 0.05).