Characterization of the nod and sdh operons in the legume symbionts Bradyrhizobium japonicum and Sinorhizobium meliloti.

摘要

This study was undertaken to characterize the nod and sdh operons of Bradyrhizobium japonicum and Sinorhizobium meliloti. Ten putative B. japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. The mutants were found to have higher nodY expression than the wild-type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1muM) and/or temperature (15°C). The expression of nodY gene induction was determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochitooligosaccharide than the wild-type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. Based on nodY gene expression and symbiotic phenotype the B. japonicum mutants were classified into three groups.; A molecular genetic approach was taken to investigate the regulation of expression of succinate dehydrogenase (SDH) in S. meliloti. The sdhCDAB genes encoding SDH were shown by RT-PCR to be co-transcribed and thus constitute an operon. The transcriptional start site and putative promoter region of the first gene in the operon, sdhC , were identified by 5'-RACE and DNA sequence analysis. Transcriptional lacZ fusions to sdhC indicated that expression of the operon is regulated by carbon source in the growth medium but not by growth phase. The highest expression of the sdh operon was observed in cells grown with acetate, arabinose and glutamate, as sole carbon sources, and the lowest expression was observed in cells grown with glucose and pyruvate as sole carbon sources.; Also presented is the isolation and characterization of the first defined sdh mutant in a rhizobial species. The mutants helped demonstrate that the total lack of SDH activity would be lethal to S. meliloti cells. Symbiotic phenotype of the mutants indicated that SDH is required for N2-fixation.