Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

Genetic diversity of root nodulating bacteria associated with Retama sphaerocarpa in sites with different soil and environmental conditions

The genetic diversity of root nodulating bacteria isolated from Retama sphaerocarpa was studied using BOX-A1R PCR and phylogenetic analysis of the 16S rRNA region, as well as the housekeeping genes atpD, glnII and recA. A total of 193 isolates were obtained from eight different sites with different soil and environmental conditions in the Iberian Peninsula. These isolates corresponded to 31 different strains that successfully nodulated R. sphaerocarpa seedlings in reinoculation trials. About one-third of the strains clustered with B. canariense or B. cytisi within Bradyrhizobium group I. The remaining strains clustered with B. elkanii/B. pachyrhizi within Bradyrhizobium group II or in separate clades that could represent new lineages. Based on the 16S rRNA and combined atpD + glnII + recA sequences, two to three lineages of root nodulating bacteria were found at each sampling site, except for Collado Garcia where five species were detected. B. canariense and B. elkanii/B. pachyrhizi were the most abundant species, whereas the least abundant were those related to B. retamae and a putative new lineage. B. canariense was found only in soils with neutral and acid pH, whereas B. retamae was the dominant species in alkaline soils.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.