**3.** *Bradyrhizobium japonicum* **and its proteomics/exoproteomics**

Culturing bacteria *in vitro* can cause changes in the bacterial physiology and genetics. In or‐ der to discriminate between types of these differences, *B. japonicum* cultivated in HM media and those isolated from root nodules were studied for their protein profile using 2-D PAGE and MALDI-TOF. The cultured cells showed greater levels of proteins related to fatty acid, nucleic acid and cell surface synthesis. While carbon metabolism proteins related to global protein synthesis, maturation and degradation and membrane transporters seemed to be similar in both cultured and nodule isolated bacteria, nitrogen metabolism was more pro‐ nounced in the bacteroids. Despite the quantitative differences in some proteins in the cul‐ tured and nodule isolated bacteria, it was observed that the various proteins in common between them performed similar functions [124]. A high resolution 2-D gel electrophoresis analysis of these bacteroids revealed a number of proteins, of which about 180 spots could be identified using the *B. japonicum* database (http://www.kazusa.or.jp/index.html) [125]. The bacteroids showed a lack of defined fatty acid and nuclei acid metabolic pathways, but were rich in proteins related to protein synthesis, scaffolding and degradation. Other pro‐ teins with high expression levels were associated with cellular detoxification, stress regula‐ tion and signalling, all of which clearly establishes that differentiation into bacteroids results in a clear shift on metabolism and expression of metabolic pathways required by the bacte‐ roids for their specialized activities [126].

Since competitiveness plays an important role in this symbiotic relationship, 2-D gel electro‐ phoresis, image and data analysis, and in-gel digestion proteomic studies, were conducted on *B. japonicum* 4534, a strain with high competitiveness, and *B. japonicum* 4222, with low competitiveness, for nodulation. When treated with diadzein, both the strains showed upregulation of proteins: 24 in *B. japonicum* 4534 and 10 in *B. japonicum* 4222. Upon treatment

with diadzein and other extracellular materials such as extracellular enzymes and polysac‐ charides involved in nodulation of the strains tested, the numbers increased to 78 (43 upregulated and 35 down-regulated) and 47 (25 up-regulated and 22 down-regulated) in these two strains. Proteins not related to nodulation were also present, and the higher number of proteins expressed by *B. japonicum* 4534 may be the reason for increased competitiveness during symbiosis [127]. Comparative studies on whole cell extracts of genistein induced and non-induced cultures of a strain used in commercial inoculants in Brazil, *B. japonicum* CPAC 15 (=SEMIA 5079), and of two genetically related strains grown *in vitro* were conducted us‐ ing 2-D gel electrophoresis followed by mass spectrometry. Some of the noteworthy pro‐ teins belonged to the cytoplasmic flagellar component FliG, periplasmic ABC transporters, proteins related to the biosynthesis of exopolysaccharides (ExoN), proteins that maintain re‐ dox state and the regulon PhyR-σEcfG, which is known to increase the competitiveness of *B. japonicum* and also help the bacteria under stress conditions, and several other hypothetical proteins [128].

*B. japonicum* utilizes the bacterial Type III secretion system (TTSS). In order for TTSS to be effective it requires a flavonoid inducer. The *tts* gene cluster of *B. japonicum* is regulated by the isoflavone genistein. In its presence NodD1 and NodW activate the *ttsI*, which is a twocomponent response regulator, necessary for expression of other genes in the *tts* cluster. In addition, the operons governing the TtsI regulon have a conserved motif in the *tts* box pro‐ motor region, which underscores the importance of regulation of TTSS in *B. japonicum*. Flag‐ ellin is a bulk protein synthesized by *B. japonicum* that plays an important role in TTSS. Mutant *B. japonicum* cells created by deleting the flagellin genes *bll6865* and *bll6866* were studied for their exoprotein profiles, in comparison with the non-mutated strains. Upon in‐ duction using genistein, it was observed that amongst the identifiable proteins, Blr1752 simi‐ lar to NopP of *Rhizobium* sp. strain NGR234, Blr1656 (GunA2) having endoglucanase activity and three other proteins having similarity to proteins of the flagellar apparatus were detect‐ ed. However, none of these proteins were detected in the mutant exoproteome, suggesting that these proteins are the products of a highly conserved *tts* box motif containing genes that encode these secreted proteins [129 and references therein].

A study of 2-D gel electrophoresis combined with MALDI-TOF MS for the identification of *B. japonicum* strains 110, BJDΔ283 and BJD567 exoproteomes revealed a high frequency of substrate-binding proteins of the ABC transporter family. Addition of genistein to the cul‐ tures altered the exoproteome; three flagellar proteins and a nodulation outer protein, Pgl, were identified. Further shotgun mass spectrometry of the genistein induced exoproteome revealed the presence of nodulation outer proteins, NopB, NopH, NopT and type III-secret‐ ed protein GunA2. Addition of diadzein or coumerstrol, instead of genistein, to the cell cul‐ ture showed a reduction in the type III-secreted protein GunA2 [130]. *B. japonicum* cell lines derived from strain SEMIA 566 are adapted to stressful environmental conditions in Brazil. They also vary in their capacity for symbiotic nitrogen fixation. A representational differ‐ ence analysis study was conducted on the strains S 370 and S 516, derived from SEMIA 566. Strain S 370 produces the nodulation outer protein P gene, which is strongly associated with the TTSS, and is also the major determinant of effective nodulation [131].

*B. japonicum* strain CPAC 15 (5SEMIA 5079) is a strain used in commercial inoculants; it be‐ longs to the same serogroup as strain USDA 123 and is used in Brazil on soybean. Both of these strains are known to be highly competitive and saprophytic. Apart from *B. japonicum* strain USDA 110, which has been sequenced [24,25], CPAC 15 is the only stain that has been partially sequenced in any significant measure [132]. CPAC 15 and two related strains, S 370 and S 516, were studied using whole-cell 2-D protein gel electrophoresis and spot profiles of selected proteins using MS. Cytoplasmic and periplasmic proteins found to occur in diverse metabolic pathways related to the saprophytic properties of CPAC 15; 26 hypothetical pro‐ teins were identified [133].

*B. japonicum* strain USDA 110 from soybean plants cultivated in growth chambers were har‐ vested at 21 days of symbiosis and subjected to transcriptomics studies and proteomics us‐ ing gelLC-MS/MS. Through this integrated approach 27.8% of the theoretical proteome and 43% of the predicted genes and proteins were detected. Analysis of the biological and func‐ tional pathways highlighted proteins involved in carbon and nitrogen metabolism: several enzymes of the TCA cycle, gluconeogenesis and pentose phosphate pathway. Experiments with bacteroids obtained from soybean plants grown under field conditions showed identi‐ cal results [134 and references therein].
