The nodulation of Glycine max cv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-type Bradyrhizobium japonicum USDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (10 9 cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (10 5 cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of an nodY-lacZ fusion, this phenomenon was attributed to bradyoxetin-induced repression of nod gene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 10 9 cells/ml of the nodD2, nolA, nodW, and nwsB mutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 10 5 cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-densitydependent nodulation of soybeans. In contrast, the number of nodules produced by an nodD1 mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. The nodD2 mutant outcompeted B. japonicum strain USDA123 for nodulation of G. max cv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that the nolA and nodD2 mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that the nodD2 mutant of B. japonicum may be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicum symbiosis.Bradyrhizobium japonicum is the nitrogen-fixing, root nodule symbiont of soybeans. Results from several studies have shown that both the bacterial and host genotypes influence the symbiotic interaction of Bradyrhizobium with soybeans (32). Several soybean genotypes, including cultivars and plant introductions (PI), were shown to be differentially nodulated by specific strains or genotypes of Bradyrhizobium japonicum (4, 28). Cregan and colleagues (4, 5) and Lohrke et al. (22) identified two soybean PI genotypes, PI 377586 and PI 417566, which restricted nodulation by B. japonicum strains in serogroups 123 and 110, respectively. The results of several subsequent studies done using one or both PI genotypes indicated that host-controlled restriction of nodulation is strain and temperature dependent, determined by the root genotype, and conditioned by a single recessive host gene (22,23). Moreover, results from preliminary microscopic studies done using both PI genotypes suggested that the nodulation restriction process occurred some time after the formation of nodule primordia in incompatible h...

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 À1 CuCl 2 . In contrast, the closely related, plasmid-free Bradyrhizobium sp. strain ORS278 could not grow at copper concentrations exceeding 100 mg l À1 . 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.