Cloning and characterization of protocatechuate 3,4-dioxygenase genes from Bradyrhizobium japonicum USDA110

Cloning of Protocatechuate 3,4-dioxygenase genes (PCD) from Bradyrhizobium japonicum USDA110 was achieved by screening a genomic library using a heterologous PCD gene probe from Pseudomonas cepacia. The cloned PCD gene from B. japonicum was characterized by enzyme assay and sequence analysis. The nucleotide sequence revealed that there are two open reading frames of 651 bp and 561 bp which encode polypeptides with predicted molecular weights of 25 kilo daltons and 20 kilo daltons. DNA sequence comparisons revealed that the PCD gene from B. japonicum USDA110 shows 51-57% sequence homology with three other PCD genes. Deduced amino acid sequence comparison revealed that the PCD from B. japonicum has diverged compared to PCD from Pseudomonas putida, Acinetobacter calcoaceticus and P. cepacia. The authenticity of the cloned B. japonicum PCD gene was confirmed by complementing P. cepacia DBO167 strain mutant for the PCD gene. The functional significance of chromosomal PCD gene of B. japonicum was determined, by construction of mutants for PCD gene and comparison of the survival of mutant with the wild B. japonicum in soil. Restriction enzyme analysis and Southern hybridization using kanamycin resistance gene probe revealed the presence of mutagen Tn5 inserted into the PCD gene. The mutant PCD gene was used to replace the chromosomal PCD gene by marker exchange technique. The verification of constructed mutants was established by testing for its inability to grow on basal media containing parahydroxybenzoic acid as sole carbon source and its ability to grow on complete medium containing kanamycin. Southern hybridizations were also performed using PCD gene probe and NPTII gene probe. Results from the above experiments showed the integration of Tn5 on the chromosome of B. japonicum and disruption of the PCD gene in the mutant B. japonicum. The survival and competition problem of B. japonicum USDA110 was studied by interacting the plant with the wild and mutant B. japonicum, and enumeration of B. japonicum from soil samples. This study revealed that PCD gene has an important role in the saprophytic survival of B. japonicum in the rhizosphere.