Effect of Dihydrolipoamide Dehydrogenase LpdA3 Gene Knockout in Sinorhizobium meliloti Metabolism

Abbas, R.; Sorour, Noha

doi:10.21608/jacb.2016.40895

Effect of Dihydrolipoamide Dehydrogenase LpdA3 Gene Knockout in Sinorhizobium meliloti Metabolism

Document Type : Original Article

Authors

R. Abbas ¹
Noha Sorour ²

¹ Department of MicrobialBiotechnology, Genetic Engineering and BiotechnologyResearch Institute, University of Sadat City, Sadat City, Egypt, 22857/79

² Departmentof IndustrialBiotechnology Genetic Engineering and BiotechnologyResearch Institute, University of Sadat City, Sadat City, Egypt, 22857/79

10.21608/jacb.2016.40895

Abstract

The genome of the symbiotic N₂-fixing soil bacterium Sinorhizobium meliloti encodes three lpdA alleles. Based on their position relative to genes encoding the other complex subunits, each of the lpdA alleles is predicted to function in a different enzyme complex. The lpdA1 is encoding the E3 component of pyruvate dehydrogenase (PDH);lpdA2, the E3 component of 2-Oxyglutarate dehydrogenase (OGD), while the lpdA3 is presumed to encode the E3 subunit of a branched-chain alpha-ketoacid dehydrogenase (BKD). To date, no functional characterization of lpdA3 gene has been done in S. meliloti. Analysis of the LpdA3 amino acid sequences revealed conserved functional domains, suggesting that the S. meliloti lpdA3 allele encode functional proteins, as well as, each may be specific to the complex encoded by the adjacent gene. To test this hypothesis, insertion mutation was induced in the lpdA3 allele. Internal fragment of lpdA3 allele cloned into plasmid pVIK112 recombined into the S. meliloti genome by single cross-over yielded lpdA3 mutant. The resulting mutant carried transcriptional lpdA:lacZ fusions. The carbon and amino acids utilization phenotype of lpdA3 mutant was found to be distinct and indicative of the enzyme complex rendered non-functional by the mutation, BKD is almost demolished in the lpdA3 mutant and both the PDH and OGD activities were similar to wild-type (WT) level.

Keywords