Cloning and characterization of a NADP+-malic enzyme gene from Bradyrhizobium japonicum USDA110

Malic enzymes have been considered to play a key role in energy metabolism for nitrogenase reaction in bacteroids. To elucidate the physiological role of the malic enzymes in Bradyrhizobium japonicum bacteroids, a putative malic enzyme gene Bjtme1 was cloned by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes and draft sequence data of the Bradyrhizobium japonicum USDA110 genome sequence project. To confirm the characteristics of the Bjtme1 gene, the protein encoded by this gene was over-expressed using a pET32a(+) system and it exhibited a NADP⁺-malic enzyme (EC 1.1.1.40) activity, indicating that Bjtme1 was the gene of the NADP⁺-malic enzyme. This is the first report on the cloning and characterization of the NADP⁺-malic enzyme gene from B. japonicum, and the gene structure was compared with that of NADP⁺-malic enzyme genes of other rhizobia.     

Estimation of 140S particles in foot-and-mouth disease virus (FMDV) vaccine by using the computer analyzing system

The quantity of 140S particles in inactivated foot-and-mouth disease virus (FMDV) vaccine samples produced in Foot-and-Mouth Disease Vaccine Production Center (FMD Vaccine Production Center) in Thailand was estimated by the sucrose gradient ultracentrifugation and optical density analysis by using the computer applying system. The soft ware; Chromato Data System (CDS) (Nihon Chromato Works Co., Ltd. Japan) which is prepared for the analysis of chromatography, was applied for the estimation of 140S particles in FMDV vaccine. The quantity of 140S particles in each vaccine sample measured by CDS was mostly ranged from 2-4 micrograms/ml and this quantity was consistent with the results of the other reports. This method is considered to be the available method for estimation of 140S particles in FMDV vaccine as routine assay.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.