Homology of nod GENES in associative nitrogen-fixing bacteria

Abstract

Klebsiella oxytoca strain R15, R17 and NG13 are nitrogen-fixing bacteria that associate on the rice root surface with secretory root lectin as mediator. Since rice lectins bind specifically with N-acetylglucosamine (GlcNAc), the carbohydrate moiety of the nodulation factor of Rhizobium meliloti, which mediate that first step of Rhizobium-legume interaction, and its induction was known to be involved with the common nodulation genes (nodDABC) in response to the plant signal, isoflavonoid. The objective of this research is to use these nodD1 ABC genes from R. meliloti to search for homology of these nod genes in the local strains of associative K. oxytoca in comparision with other associative Azospirillum brasilense Sp7 and free-living K. pneumonia strain M5al, by using rhizobia strains as references. Plasmids containing nifHDK genes, from K. pneumonia M5al was also used as probe to study nod and nif organization. After labeling with DIG-DNA labeling kit, Southern hybridization was performed with digested chromosomal DNA from these N₂-fixing bacteria. The BamHI digested DNA from associative K. oxytoca strain R15, R17 and NG13 show two DNA fragments of 4.0 and 4.9 kb that hybridize with nodD1 probe, which resemble other rhizobia strains and associative A. brasilense Sp7, except the free-living K. pneumonia M5al. However, there was no DNA fragment that were homologous with nodAB and nodC, and no evidence for closely related organization between nifHDK and nodD1 by restriction fragment length polymorphism. The nodD1-liked gene was therefore cloned from strain NG13 and harboured in pUC18m resulted in two recombinant plasmids: R1 and R2 carrying 4.0 and 4.9 kb of BamHI internal fragment hybridized with nodD1 probe. In addition, the results indicate the potential of using nod and nif probes in the identification of rhizobia.