Restriction fragment length polymorphism (Rflp) of nitrogen-fixing bacteria associated with rice (Oryza sativa L.)

Abstract

Klebsiella R15 and R17 are associative nitrogen-fixing bacteria in the rhizosphere of rice. In associative rice-Klebsiella system, the nitrogenase activity is serveral hundred folds higher than free-living condition. The aim of this research is to compare the restriction fragment length polymorphism (RFLP) between the associative Klebsiella strains R15 and R17 with the best known free-living K. pneumoniae M5al by using restriction enzymes and Southern hybridization with nif structural genes and glnA gene of K. pneumoniae M5al as probes. Since both Klebsiella strains R15 and R17 have no plasmids, their chromosomal DNAs were purified and digested individually with BamHI, BglII, EcoRI, HindIII, PstI, SalI, SmaI and XhoI for the RFLP patterns to compare with those of associative K. oxytoca NG13, the rhizospheric diazotroph isolated from japonica rice and free-living K. pneumoniae M5a1. Restriction patterns of all restriction enzymes show pattern of discrete bands which can distinguish the 3 associative Klebsiella strains from the free-living K.pneumoniae M5al, but there is no polymorphism among the 3 associative strains. Southern hybridization between pSA30 containing nif structural genes region and digested DNA show identical profile of hybridization band among 3 associative strains NG13, R15 and R17, which differ from freeliving M5a1 only in DNA subjected to BglII, PstI and SmaI digestion. Probing with subfragments Fa, Fb and Fc of nifHDKTYE containing nifHD, nifDK and nifKTYE respectively locate the differences in BglII, PstI and SmaI restriction sites from the restriction map of K. pneumoniae to reside in nifL, nifE and nifJ regions respectively. Southern hybridization of DNA, digested with 7 restriction enzymes which cut within glnA ntrBC region, with glnA probe also shows high degree of homology among all Klebsiella strains. The 3 associatve strains differ from free-living M5al only by XhoI cutting which is outside the glnA ntrBC region. All these results indicate coevolution of the 3 associative Klebsiella NG13, R15 and R17 with highly conserved nif genes region, and glnA ntrBC region; only a few changes in DNA sequences from the strictly free-living K. pneumoniae M5al can be detected.