Importance of trehalose biosynthesis for Sinorhizobium meliloti Osmotolerance and nodulation of Alfalfa roots

Abstract

The disaccharide trehalose is a well-known osmoprotectant, and trehalose accumulation through de novo biosynthesis is a common response of bacteria to abiotic stress. In this study, we have investigated the role of endogenous trehalose synthesis in the osmotolerance of Sinorhizobium meliloti. Genes coding for three possible trehalose synthesis pathways are present in the genome of S. meliloti 1021: OtsA, TreYZ, and TreS. Among these, OtsA has a major role in trehalose accumulation under all of the conditions tested and is the main system involved in osmoadaptation. Nevertheless, the other two systems are also important for growth in hyperosmotic medium. Genes for the three pathways are transcriptionally responsive to osmotic stress. The presence of at least one functional trehalose biosynthesis pathway is required for optimal competitiveness of S. meliloti to nodulate alfalfa roots.

FIG. 1.

Construction of S. meliloti 1021 otsA (a), treY (b), and treS (c) mutants. The wild-type version of the amplified region obtained by PCR is represented, and the mutated version is shown below. E, EcoRI; M, MluI; EV, EcoRV; S, SmaI; H, HindIII; Xh, XhoI; Km^R, kanamycin resistance cassette; Sm^R/Spc^R, streptomycin/spectinomycin resistance cassette.

FIG. 2.

Growth curves of S. meliloti 1021-derived strains in MM (a), MM supplemented with 0.7 M sucrose (b), and MM supplemented with 0.5 M NaCl (c). Only the growth curves of mutants showing significant differences with the wild-type strain are shown. Wild-type strain 1021, squares; TreS⁻ OtsA⁻ mutant 10trOt, triangles; TreS⁻ TreY⁻ OtsA⁻ mutant 10SYOt, circles. The data shown are representative of at least two independent experiments. OD600, optical density at 600 nm.

FIG. 3.

Ability of 1021-derived mutants to grow in MM with 0.5 M NaCl added at pH 7 (a) and pH 6.5 (b). In each row, drops contain approximately the number of CFU indicated at the left. A representative example of at least two experiments is shown. Pictures were taken after 6 to 8 days of incubation under the indicated conditions.

FIG. 4.

Growth curves of strains 1021 (wild type; squares) and 10SYOt (TreS⁻ TreY⁻ OtsA⁻ mutant; circles) in MM with NaCl added at the times indicated by the arrows to a final concentration of 0 M (a) or 0.6 M (b). The data shown are representative of at least two independent experiments. OD600, optical density at 600 nm.

FIG. 5.

Expression of the transcriptional fusions otsA::uidA (a), treY::uidA (b), and treS::uidA (c) during growth in MM (left) or MM plus 0.5 M NaCl (right). The data shown are representative of two independent experiments. OD600, optical density at 600 nm; Miller U, β-glucuronidase activity expressed as Miller units.

FIG. 6.

Trehalose accumulation by S. meliloti 1021 during growth in MM (a) and by 1021-derived mutants at the late stationary phase of growth (70 h after inoculation) (b). OD600, optical density at 600 nm. Bars correspond to standard errors, and different letters indicate significant differences according to an ANOVA test (P < 0.01). The data are representative of two independent experiments.

FIG. 7.

Trehalose accumulation by S. meliloti 1021 after an osmotic shock caused by the addition of different NaCl concentrations to mid-exponential-phase cultures growing in MM (a) and by 1021-derived mutants after an osmotic shock with 0.3 M NaCl (b). Cells were harvested 24 h after the saline shock. Bars correspond to standard errors, and different letters indicate significant differences according to an ANOVA test (P < 0.01). The data shown are representative of two independent experiments.

FIG. 8.

Nodule competition assays. The data shown represent the percentages of nodules occupied by the indicated strain after coinoculation (ratio 1:1) with strain 1021 (pGUS3). Bars correspond to standard errors, and different letters indicate significant differences according to an ANOVA test (P < 0.05). The data shown are representative of two independent experiments.