Transcriptional and physiological responses of Bradyrhizobium japonicum to desiccation-induced stress

Abstract

The growth and persistence of rhizobia and bradyrhizobia in soils are negatively impacted by drought conditions. In this study, we used genome-wide transcriptional analyses to obtain a comprehensive understanding of the response of Bradyrhizobium japonicum to drought. Desiccation of cells resulted in the differential expression of 15 to 20% of the 8,453 [corrected] B. japonicum open reading frames, with considerable differentiation between early (after 4 h) and late (after 24 and 72 h) expressed genes. While 225 genes were universally up-regulated at all three incubation times in response to desiccation, an additional 43 and 403 up-regulated genes were common to the 4/24- and 24/72-h incubation times, respectively. Desiccating conditions resulted in the significant induction (>2.0-fold) of the trehalose-6-phosphate synthetase (otsA), trehalose-6-phosphate phosphatase (otsB), and trehalose synthase (treS) genes, which encode two of the three trehalose synthesis pathways found in B. japonicum. Gene induction was correlated with an elevated intracellular concentration of trehalose and increased activity of trehalose-6-phosphate synthetase, collectively supporting the hypothesis that this disaccharide plays a prominent and important role in promoting desiccation tolerance in B. japonicum. Microarray data also indicated that sigma(54)- and sigma(24)-associated transcriptional regulators and genes encoding isocitrate lyase, oxidative stress responses, the synthesis and transport of exopolysaccharides, heat shock response proteins, enzymes for the modification and repair of nucleic acids, and the synthesis of pili and flagella are also involved in the response of B. japonicum to desiccation. Polyethylene glycol-generated osmotic stress induced significantly fewer genes than those transcriptionally activated by desiccation. However, 67 genes were commonly induced under both conditions. Taken together, these results suggest that B. japonicum directly responds to desiccation by adapting to changes imparted by reduced water activity, such as the synthesis of trehalose and polysaccharides and, secondarily, by the induction of a wide variety of proteins involved in protection of the cell membrane, repair of DNA damage, stability and integrity of proteins, and oxidative stress responses.

FIG. 1.

Viability of B. japonicum cells following incubation under desiccating (27% RH) and hydrated (100% RH) conditions. (A) Plate count data expressed as log₁₀ CFU/ml for desiccated and hydrated cells at the 4- and 72-h incubation times. Error bars representing the standard deviation of three biological replicate samples are shown. (B) Percentages of live cells obtained by Baclight following 4-, 24-, and 72-h incubation periods.

FIG. 2.

Venn diagrams showing statistically significant (α = 0.05) up- and down-regulated genes in the desiccated versus hydrated treatments from B. japonicum microarray analyses. Values shown represent more than 2- and 1.5-fold (in parentheses) differential expression. Identified genes had a global false discovery rate of ≤5%. Six arrays representing a total of 12 replicates of each ORF were analyzed for each of the three desiccation times.

FIG. 3.

Functional categories of statistically significant (α = 0.05), differentially expressed genes. (A) Genes expressed only in the early (4-h) desiccation period. (B) Genes commonly expressed in the 24- and 72-h desiccation periods. Black bars, up-regulated genes; gray bars, down-regulated genes.

FIG. 4.

qRT-PCR analysis of differentially expressed trehalose synthesis genes (otsA, treS, treY) in desiccated and hydrated B. japonicum cells normalized to the housekeeping gene parA. (A) Ratio of trehalose synthesis gene expression in desiccated versus hydrated samples. (B) Absolute expression of trehalose synthesis genes (otsA, treS, treY) in desiccated B. japonicum cells (values for treY relative to parA were <0.01; thus, the graph bars are difficult to see). Black bars, otsA; gray bars, treS; white bars, treY. Error bars representing the standard deviation of three biological replicates samples are shown.

FIG. 5.

Intracellular trehalose concentrations in desiccated and hydrated B. japonicum cells. Values are normalized based on cell viability determined by using LIVE/DEAD staining. Black bars, desiccated cells (27% RH); gray bars, hydrated cells (100% RH).

FIG. 6.

Survival of B. japonicum rpoN mutant strains, relative to wild-type cells, following incubation under desiccating (27% RH) conditions. The significance of data was determined by comparison of means with ANOVA and Tukey-Kramer HSD. Columns followed by different letters are significantly different at P of 0.05. Survival rates of all strains at 100% RH were not significantly different (P < 0.001).