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.Bradyrhizobium japonicum, which belongs to the family Bradyrhizobiaceae of the order Rhizobiales, is an inhabitant of the soil which can exist in either a free-living state or an endosymbiont state in association with host plants. In the latter state, the host specificity of B. japonicum allows it to establish a symbiotic relationship with the soybean plant (Glycine max) by engaging in specific signaling and communication which involves plant-borne flavonoids and bacterial lipochitooligosaccharide Nod factors (11,40). In addition, B. japonicum is capable of diversity in its metabolism and energy production in response to environmental cues (24). One variable that changes as B. japonicum transforms from a free-living state to a symbiotic state is the concentration of oxygen. It has been demonstrated that a concentration difference on the magnitude of 5 orders of 10 (34) exists between the two states. Reactive oxygen species (ROS) are created within cellular systems as a by-product of aerobic respiration because of the incomplete reduction of molecular oxygen at the terminus of the electron transport chain. These reactive oxygen molecules are capable of modifying cellular components, such as lipids, proteins, and nucleic acids, due to their contribution to the p...

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

“…In the particular example of B. diazoefficiens, the respiration rate might be just such a signal linked to the transcription of the lateral-flagellar regulon. Previous reports indicated that situations diminishing the respiration rate, such as microaerobiosis, the bacteroid state (23), or iron deficiency (24), downregulate the lateral-flagellar regulon transcription, whereas situations known to increase the oxygen consumption, such as permanent exposure to moderate oxidative stress (25) or the use of arabinose as the sole carbon source (43; C. Cogo, J. Pérez-Giménez, C. B. Rajeswari, M. F. Luna, and A. R. Lodeiro, unpublished data), promote that transcription. Moreover, these changes in transcription were not observed in the subpolar-flagellar regulon, indicating that these stimuli act specifically on the lateral flagella.…”

“…Operon I contains flbT L , whose locus, upon activation, acts as a translation (Tln) inducer of the monocistronic lafA2 and appears to stabilize (Stb) the lafA1 transcript from a promoter within operon II. In addition to the effects of arabinose and mannitol, evidence from the literature indicates that prolonged exposure to moderate oxidative stress also induces lafR and the lateral flagellar regulon (25), whereas situations of O 2 limitation as the bacteroid state (23) or iron limitation (24) repress them. We also observed that viscosity and tortuousity of the medium induce lateral flagella (20) and that microoxia was reported as inhibiting lateral flagella genes expression (23).…”

“…Hence, growth in liquid medium with mannitol as the carbon source does not permit the expression of the lateral flagella. Transcriptomic studies have also indicated that conditions of microoxia (23) or iron deficiency (24) prevent the expression of lateralflagellar genes, whereas permanent exposure to moderate oxidative stress induces these loci (25). In the example of Ensifer meliloti and its close relative Brucella melitensis, flagellar expression is also under strict control by specific nutritional, physiological, and population-size requirements (26)(27)(28)(29).…”

DNA Microarray-Based Identification of Genes Regulated by NtrC in Bradyrhizobium japonicum

“…B. japonicum strains were cultured in 20 ml of MMB glutamate or MMB glutamate plus ammonium to an OD 600 of 0.4 to 0.6. Cells were harvested and RNA was extracted as described previously (26)(27)(28). Synthesis of cDNA and quantitative reverse transcription-PCR (qRT-PCR) was performed using 3 g of total RNA as described previously (26)(27)(28).…”

“…Cells were harvested and RNA was extracted as described previously (26)(27)(28). Synthesis of cDNA and quantitative reverse transcription-PCR (qRT-PCR) was performed using 3 g of total RNA as described previously (26)(27)(28). Data for three independent biological replicates were generated and normalized to the expression of the bll7457 gene, encoding the histidyl-tRNA synthetase HisS.…”