Genome-wide transcriptional and physiological responses of Bradyrhizobium japonicum to paraquat-mediated oxidative stress

Abstract

The rhizobial bacterium Bradyrhizobium japonicum functions as a nitrogen-fixing symbiont of the soybean plant (Glycine max). Plants are capable of producing an oxidative burst, a rapid proliferation of reactive oxygen species (ROS), as a defense mechanism against pathogenic and symbiotic bacteria. Therefore, B. japonicum must be able to resist such a defense mechanism to initiate nodulation. In this study, paraquat, a known superoxide radical-inducing agent, was used to investigate this response. Genome-wide transcriptional profiles were created for both prolonged exposure (PE) and fulminant shock (FS) conditions. These profiles revealed that 190 and 86 genes were up- and downregulated for the former condition, and that 299 and 105 genes were up- and downregulated for the latter condition, respectively (>2.0-fold; P < 0.05). Many genes within putative operons for F(0)F(1)-ATP synthase, chemotaxis, transport, and ribosomal proteins were upregulated during PE. The transcriptional profile for the FS condition strangely resembled that of a bacteroid condition, including the FixK(2) transcription factor and most of its response elements. However, genes encoding canonical ROS scavenging enzymes, such as superoxide dismutase and catalase, were not detected, suggesting constitutive expression of those genes by endogenous ROS. Various physiological tests, including exopolysaccharide (EPS), cellular protein, and motility characterization, were performed to corroborate the gene expression data. The results suggest that B. japonicum responds to tolerable oxidative stress during PE through enhanced motility, increased translational activity, and EPS production, in addition to the expression of genes involved in global stress responses, such as chaperones and sigma factors.

Fig. 1.

Effect of paraquat on the growth of B. japonicum USDA 110 in AG media. The raw absorbance data have been log₁₀ transformed and plotted as a function of time in hours. Control cultures were treated with sterilized double-distilled water (ddH₂O). Symbols are as follows: control (●); 10 μM (○); 50 μM (▾); 100 μM (▵); and 1 mM (■). Each point is the mean of three replicates, and the error bars represent the standard errors of the means.

Fig. 2.

Effect of fulminant shock (FS) treatment with paraquat on the survival of B. japonicum USDA 110. Control cultures were treated with sterilized ddH₂O. Symbols are as follows: control (●), 100 μM (○), 250 μM (▾), 500 μM (▵), 750 μM (■), 1 mM (□), 5 mM (♦), 10 mM (♢), and 20 mM (▴). Values are means ± standard errors of the means for three replications.

Fig. 3.

Comparison of the total number of differentially expressed genes between prolonged exposure (PE) and fulminant shock (FS) conditions. Values shown represent more than 2-fold (in bold) and 1.5-fold (in parentheses) differential expression with P < 0.05.

Fig. 4.

Measurement of ROS in B. japonicum cultures. Absorbance at 300 nm of 2-hydroxyethidium was calculated for each condition at the time of cell harvest. The contribution of paraquat and the media to the absorbance values were accounted for by including them as controls and subtracting those values from the measured absorbance values, resulting in relative absorbance. Black bars represent control without treatments and gray bars represent paraquat treatment conditions (100 μM for prolonged exposure [PE] and 5 mM for fulminant shock [FS]). The differences between control and treatment for both treatments are statistically significant (P < 0.05). Error bars represent the standard errors of the means for nine replicates.

Fig. 5.

Functional classification of differentially expressed genes with >2.0-fold changes. (A) Comparison of differential expression under the prolonged exposure (PE) condition. (B) Comparison of differential expression under the fulminant shock (FS) condition. Black bars represent positive fold induction values and gray bars represent negative fold induction values. Functional classifications were derived from B. japonicum genome annotations available through Rhizobase (http://bacteria.kazusa.or.jp/rhizobase/).

Fig. 6.

Comparison of log₂-transformed qRT-PCR data and microarray data of 18 representative genes from both conditions. These genes were selected based on fold induction and functional categories. The corresponding gene names for the locus IDs are as follows: bll1028 (carQ), blr2764 (fixO), blr4635 (groEL), bll5813 (flgC), blr2221 (bioA), bll6866 (fla), blr6333 (bkdB), bll5412 (rplJ), bll6879 (fliN), and blr2581 (cbbF). blr1602, blr4258, bll0256, blr3815, bll4012, bsl2574, blr4701, blr5517, and bll0631 lack assigned names. Open triangles, prolonged exposure (PE); closed triangles, fulminant shock (FS).

Fig. 7.

Ratio of treatment (100 μM paraquat) to control of relative CFU in capillaries plotted against time. Error bars are the standard errors of the means for three replicates. If the ratio is >1, chemotaxis is positive and the test substance is an attractant. If the ratio is <1, chemotaxis is negative and the test substance is a repellant. A ratio close to 1 is indicative of no net effect on chemotaxis. Symbols are as follows: wild type (○) and cheA mutant (▿).

Fig. 8.

Quantification of the total EPS (A) and acidic sugar content (B) isolated from the supernatant of B. japonicum cultures treated with 0, 100, 250, and 500 μM paraquat. All values in the paraquat treatments are statistically significant from control (P < 0.05). Error bars represent the standard errors of the means for three replicates.