Characterization of Bacteroid Proteins in Soybean Nodules Formed with Bradyrhizobium japonicum USDA110

Abstract

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins of Bradyrhizobium japonicum USDA110 in the symbiotic and non-symbiotic state. When proteome maps were compared and the characterization of bacteroid-specific proteins was performed by N-terminal amino acid sequencing and matrixassisted desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, putative identity was assigned to 61 bacteroid protein spots, including many metabolic proteins and ABC transporters as well as nitrogenase proteins like NifH. This study shows that proteome analysis will be a useful tool for surveying genes contributing to rhizobial functions in symbiosis.Key words: 2-D gel electrophoresis, Bradyrhizobium japonicum USDA110, MALDI-TOF mass spectrometry, nodule bacteroid, symbiosisSoil bacteria, rhizobia, can form a symbiotic tissue (nodule) in the root of leguminous plants and differentiate to fix nitrogen using photosynthate as an energy source. Since this reaction to produce ammonia from nitrogen gas in the air is useful for agriculture, various researchers have attempted to introduce this system into other crop plants, and genome projects including proteomics have been applied to this complex system to elucidate the molecular mechanism inducing this organogenesis. The proteomic analysis of cultured cells and bacteroids in Sinorhizobium meliloti has provided insight into how microsymbionts alter metabolism to establish an endo-symbiotic relationship with the host plant cells 10) . In soybean, the genomic DNA of Bradyrhizobium japonicum USDA110 has been completely sequenced by the Kazusa DNA Research Institute 5) , and a large scale macroarray analysis is being conducted by comparing gene expression profiles among rhizosphere cells, cultured cells and bacteroids (Minamisawa et al., personal communication). In cooperation with these transcriptome analyses, we compared protein profiles between bacteroids and cultured cells of B. japonicum USDA110 and examined the physiological role of bacteroid specific proteins.Soybean (Glycine max L. Merr. cv. Akishirome) seeds were inoculated with Bradyrhizobium japonicum USDA110 and germinated on moist vermiculite. The seedlings (7-10 days old) were grown hydroponically in a nitrogen-free nutrient solution 17) . Nodules were harvested from plants 4 to 5 weeks after inoculation when the acetylene reduction activity per gram fresh weight of nodules reached a maximum 17) .Bacteroids were isolated from nodules as reported by Day et al. 3) , and were monitored with marker enzyme assays; cytochrome c oxidase for mitochondria 16) , alcohol dehydrogenase for cytosol 15) and b-hydroxybutyrate dehydrogenase (b-HBDH) for bacteroids 14) . High-level b-HBDH

Biogenic Amines in Rhizobia and Legume Root Nodules

“…Genetic studies to delineate the precise biosynthetic and regulatory mechanisms for biogenic amines in rhizobia and legume root nodules are required. The characterization of bacteroid proteins has recently been reported (65) and showed the usefulness of proteomics for surveying genes contributing to rhizobial functions. Such studies might help to elucidate the molecular basis underlying the various physiological and biochemical processes involved in rhizobialegume symbiotic interactions.…”

Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator GenenifA2inMesorhizobium lotiMAFF303099

“…The other effect is a positive effect on the establishment and/or maintenance of mature nodules (7,23). In Glycine max, ACC deaminase protein was reported to accumulate in the bacteroid (12). In canola roots, ACC deaminase-producing E. cloacae changed the expression of host genes involved in the defense response and cell division (14).…”

Genomes of the Symbiotic Nitrogen-Fixing Bacteria of Legumes

“…Several proteomic analyses have now provided additional insight into the symbiosis between S. meliloti and B. japonicum and their host legumes (Natera et al, 2000;Hoa et al, 2004;Emerich, 2005, 2006). One trend that has emerged during proteomic studies of B. japonicum and S. meliloti is the preferential expression of a subset of ATP-binding cassette-type transporters in nodule bacteria (Djordjevic, 2004;Hoa et al, 2004;Sarma and Emerich, 2006).…”

“…One trend that has emerged during proteomic studies of B. japonicum and S. meliloti is the preferential expression of a subset of ATP-binding cassette-type transporters in nodule bacteria (Djordjevic, 2004;Hoa et al, 2004;Sarma and Emerich, 2006). Free-living cells of both species express a large number of sugar transporters and expression of these systems is down-regulated in planta (Djordjevic, 2004;Sarma and Emerich, 2006).…”

“…The transcriptomes and proteomes of bacteroids differ considerably from those of free-living cells (Perret et al, 1999;Natera et al, 2000;Ampe et al, 2003;Barnett et al, 2004;Becker et al, 2004;Djordjevic, 2004;Hoa et al, 2004). Overall, gene expression is largely down-regulated in bacteroids (Barnett et al, 2004;Becker et al, 2004;Capela et al, 2006), possibly as a result of growth arrest and a stationary phase-like existence (Capela et al, 2006).…”