**4. Other dimensions to soybean-rhizobacteria interactions**

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

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

*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

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

encode these secreted proteins [129 and references therein].

the TTSS, and is also the major determinant of effective nodulation [131].

proteins [128].

Relationships

16

Apart from *B. japonicum*, which produces LCOs, other rhizobacteria, such as *Bacillus thurin‐ giensis* NEB17 reside in the rhizosphere of higher plants [135], forming a phyto-microbiome, much like the human microbiome, now realized to be so important in human health [136]. *Bacillus thuringiensis* NEB17 is symbiotic with *B. japonicum*, produce bacteriocins. *Bacillus* species were first reported to produce bacteriocins in 1976. The low-molecular-weight bac‐ teriocins of gram-positive bacteria have bactericidal activity, mainly against certain other gram-positive bacteria [137]. Bacteriocins are ribosomally produced peptides which affect the growth of related bacterial species. The most studied bacteriocin is colicin, produced by members of the Enterobacteriaceae [138]. Due to their commercial importance as natural preservatives and as therapeutic agents against pathogenic bacteria, these antimicrobial peptides have been a major area of scientific research [137,139].

Bacteriocins are grouped into four distinct classes based on the peptide characteristics such as post translational modifications, side chains, heat stability, N-terminal sequence homolo‐ gy and molecular weight [140]. *Bacillus thuringiensis* NEB17 was isolated from soybean root nodules as putative endophytic bacteria in 1998 in our laboratory. When co-inoculated with *B. japonicum* under nitrogen free conditions this bacterium promoted soybean growth, nodu‐ lation and grain yield [141, 142]. Subsequently, the causative agent of plant growth promo‐ tion, a bacteriocin, was isolated from *B. thuringiensis* NEB17, and is now referred to as thuricin 17 [143]. Initially, its partial sequence was determined [144], and its full sequence has been more recently reported [145]. Thuricin 17 is a low molecular weight peptide of 3162 Da, stable across a pH range of 1.0–9.25, highly heat resistant and is inactivated by treatment

with proteolytic enzymes. Based on its N-terminal sequence homology of thuricin 17 and that of the also newly isolated bacthuricin F4, a new class of bacteriocins, class IId was pro‐ posed [143]. The bacteriocins produced by *B. thuringiensis* strain NEB17 (Th17) and *B. thurin‐ giensis* subsp. kurstaki BUPM4 (bacthuricin F4 - 3160.05 Da) have been reported to show functional similarities and anti-microbial activities [146]. In addition, thuricin 17, applied as leaf spray and root drench, has positive effects on soybean and corn growth, which was first reported from our laboratory [145]; this constituted the first report of plant growth stimula‐ tion by a bacteriocin.

Proteomic profiling of both these bacteria are underway in our laboratory and we hope to acquire some indications of plant proteomic shifts related to biological nitrogen fixation through these experiments over the next few months.
