Our objectives are to characterize the role of bacterium-bacterium signaling in survivability, competitiveness and nitrogen fixation of inoculant strains. One of the major drawbacks to commercial inoculant strains is the inability to compete with native strains that are less efficient at nitrogen fixation. Factors that may influence this competitiveness include: survivability of the inoculant, competition with other bacteria for binding sites on the surface of the root and appropriate bacterium-bacterium signaling to monitor the infection process. Loh et al have shown that nod genes are down-regulated by bradyoxetin, which is produced at high cell densities, such as those attained during the cultivation of inoculant strains. This demonstrates that density-dependent phenomena can influence nodulation. They have also demonstrated that mutants that are unable to produce bradyoxetin can out compete wild-type strains for nodulation, however, the resulting constitutive expression of nod genes in the nodule result in a nitrogen fixation deficiency. Our laboratory has recently initiated a new research effort to examine quorum-sensing phenomena in B. japonicum and currently has funding from the Missouri Research Board and US Department of Agriculture to characterize the AHL-like signal molecules produced by B. japonicum strain 61A227. We are also interested in determining if a correlation exists between the competitiveness of various inoculant strains and AHL production. We are currently cloning the genes likely to be responsible for AHL production and gene regulation in B. japonicum. These cloned genes will be used to make strains unable to produce or respond to these signal molecules. This project is critical for determining the role of bacterium-bacterium signaling in competitiveness and survivability. In addition, the products of this project will provide the tools to begin dissecting the regulatory networks controlling the transition from free-living bacterium to symbiotic bacteroid. AHLs have been detected in Rhizobium leguminosarum, Rhizobium etli, and Rhizobium meliloti and in many cases, multiple AHL molecules are detected. In R. leguminosarum, AHLs are required for activation of the rhiABC operon (a set of rhizosphere-expressed genes), raiIR and cinIR genes and is involved in root nodulation and growth inhibition. Until recently, AHL autoinducers had not been detected in B. japonicum the symbiont of soybean, a major agricultural crop. In the next section we present evidence for the production of an N-acyl homoserine lactone AI by several strains of B. japonicum Using the NTL4/pZLR4 indicator strain described by Piper et al we screened twenty-three strains of B. japonicum for production of AHL autoinducer(s). Agrobacterium strain NTL4 does not produce AHL and the plasmid pZLR4 contains the Agrobacterium traR gene (required for AHL dependent gene regulation) and a gene fusion between the Agrobacterium traG gene and the lacZ gene from Escherichia coli. TraR induction of the traG::lacZ gene fusion requires AHL; therefore, this strain does not express the traG::lacZ fusion unless provided exogenous AHL. Culture supernatants from AHL producing strains are capable of inducing traG::lacZ when added to cultures of the NTL4/pZLR4 indicator strain.



Culture supernatants of eight of the twenty-three B. japonicum strains tested (61A118b, 61A224, 61A227, NRRL B-4350, NRRL B-14143, NRRL B-14080, NRRL B-14483 and NRRL L-241) were found to produce AHLs or AHL-like molecules. To our knowledge, this was the first evidence of AHL or AHL-like autoinducer production in B. japonicum capable of inducing expression of the traG::lacZ gene fusion. One of the B. japonicum strains tested and found to not produce detectable quantities of AHL was USDA 110. Lack of detectable AHL production in culture supernatants of this common laboratory strain may explain why AHLs were not previously detected in B. japonicum. (The strains in which AHL molecules were detected in B. japonicum are commercial inoculant strains).