Functional role of Bradyrhizobium japonicum trehalose biosynthesis and metabolism genes during physiological stress and nodulation.

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple DeltaotsA, DeltaotsA DeltatreS and DeltaotsA DeltatreY, and DeltaotsA DeltatreS DeltatreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.

Insights learned from pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1 and prevalence of accessory plasmids in other Bradyrhizobium sp. strains.

In silico, physiological and in planta analyses were used to characterize pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1, and assess its potential ecological function under free-living and symbiotic growth conditions. Sequence analysis revealed the presence of an uptake hydrogenase system, a repABC family plasmid replication module and open reading frames encoding type IV secretion system, TraI and TraR autoinducer proteins and several copper resistance-related proteins. Bradyrhizobium sp. BTAi1 was capable of growing in 200 mg l(-1) CuCl2. In contrast, the closely related, plasmid-free Bradyrhizobium sp. strain ORS278 could not grow at copper concentrations exceeding 100 mg l(-1). The plasmid-localized hydrogenase genes were phylogenetically distinct from those typically found in other rhizobial species, and were most related to hup genes from Thiobacillus denitrificans. The induction of the plasmid-borne hydrogenase genes during symbiosis was significantly lower than the two chromosomal-borne hydrogenase clusters. CHEF-pulsed-field gel electrophoresis was used for a comprehensive analysis of the diversity, abundance and genetic composition of accessory plasmids in other Bradyrhizobium strains. Plasmids were detected in 11 of 46 (23.9%) geographically diverse Bradyrhizobium japonicum and Bradyrhizobium elkanii strains, isolated from the United States, China and Thailand. Plasmid size was heterogeneous, ranging from 75 to 330 kb, with only two strains (DASA01244 and DASA01265) harboring plasmids with identical (240 kb) size. None of the plasmids harbored nodulation or hydrogenase genes. Taken together, our results indicate that while plasmids having ecologically significant functions may be detected in Bradyrhizobium sp. strains, they lack genes necessary for symbioses with legumes.

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

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.