Cloning, Sequencing and Expression of galE Gene of Bradyrhizobium japonicum: galE Knock-Out Mutant is Defective in Both LPS Synthesis and Nodulation of Soybean.

초록

The enzyme UDP-galactose 4-epimerase (GalE) is involved in one of the major steps of galactose metabolism in bacteria. GalE mediates the incorporation of galactose in extracellular polysaccharide materials such as the O-side chain of lipopolysaccharide (LPS). In this study, we describe the cloning and characterization of the galE gene from Brdayrhizobium japonicum, a soybean endosymbiont. Nucleotide sequence analysis of the cloned DNA identified the galE gene: Comparison of the deduced amino acid sequence with published data showed a significant homology with the GalE of Azospirillum brasilense (68%), Aquifex aeolicus (68%), and Synechocystis sp. (66%). Functional identity was achieved by the complementation of a galE mutant strain of Escherichia coli PL2 with the subcloned genes. Galactose epimerase activity of the complemented strain was essentially identical to that of the wild type E. coli DH5α. In vivo expression study showed that a 36 kDa protein was expressed from the complementing plasmids. To study the role of galE gene in B. japonicum LPS biosynthesis, the galE gene was inactivated in chromosome by double cross-over homologous recombination where galE knock-out fragment replaced the galE gene in B. japonicum. To confirm the inactivation of the galE gene in chromosomal DNA, genomic Southern blot hybridization was performed. A galE knock-out mutant strain of B. japonicum was found to be far more hydrophobic than the wild type strain based on the cell surface hydrophobicity (CSH). SDS-PAGE analysis of LPS from the galE knock-out mutant showed an LPS profile completely devoid of O-antigenic part. A standard plant infection test using the wild type and galE defective B. japonicum strains revealed that the galE gene is indeed involved in nodulation process of B. japonicum with its soybean host plant.