FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE LPDA2 GENE IN SINORHIZOBIUM MELILOTI | ||||
Menoufia Journal of Agricultural Biotechnology | ||||
Article 1, Volume 1, Issue 1, July and August 2016, Page 23-35 PDF (952.49 K) | ||||
Document Type: original papers | ||||
DOI: 10.21608/mjab.2016.176594 | ||||
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Authors | ||||
Rateb N. Abbas1; Noha M. Sorour2 | ||||
1Department of Microbial Biotechnology University of Sadat City, Sadat City, Egypt | ||||
2Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt | ||||
Abstract | ||||
Escherichia coli and many microorganisms, have a single gene encoding dihydrolipoamide dehydrogenase, which can function as the E3 subunit dihydrolipoamide (LpdA) component of different multi-enzyme dehydrogenase complexes. In contrast, Sinorhizobium meliloti genome encodes three lpdA alleles, each of the lpdA alleles are predicted to function in a different enzyme complex. The lpdA1 encode the E3 component of pyruvate dehydrogenase (PDH); while the lpdA2 is presumed to encode the E3 component of 2-Oxoglutarate dehydrogenase (OGD) and lpdA3, probable the E3 component of a branched-chain alpha-ketoacid dehydrogenase (BKD). To date, no functional characterization of the lpdA2 and lpdA3 genes has been done in S. meliloti. Analysis of the LpdA amino acid sequences revealed conserved functional domains, suggesting that the S. meliloti lpdA2 allele encode functional protein, which may be specific to the complex of E3 subunit of OGD. To test this hypothesis, insertion mutation was isolated for the lpdA2 allele. Internal fragment of lpdA2 allele was cloned into the plasmid pTH1703 and recombined into the S. meliloti genome by single cross-over which yielded lpdA2 mutant. Results obtained revealed that the LpdA2 mutated strain had greatly diminished OGD activity and wild-type levels of PDH and BKD. | ||||
Keywords | ||||
Sinorhizobium meliloti; lpdA2; TCA cycle; 2-Oxoglutarate Dehydrogenase; Mutant | ||||
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