Isolation and characterization of the Rickettsia prowazekii gene encoding the flavoprotein subunit of succinate dehydrogenase
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Z Aliabadi1, H H Winkler, D O Wood
1Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile 36688.
Abstract:
The gene (sdhA) coding for the flavoprotein subunit (SdhA) of succinate dehydrogenase of the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated using an oligodeoxyribonucleotide probe to the conserved flavin adenine dinucleotide (FAD)-binding region of characterized flavoproteins. Nucleotide (nt) sequence analysis revealed an open reading frame (ORF) of 1791 bp capable of encoding a protein of 596 amino acids (aa) with a deduced M(r) of 65,444. The deduced aa sequence, when compared to the flavoprotein subunits of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Bos taurus, revealed 52.8, 34.0, 65.8 and 52.0% aa identity, respectively. R. prowazekii SdhA produced in E. coli minicells and analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) migrated as a protein of approximately 63 kDa, comparable to the size of the deduced protein. In addition, two proteins of approximately 12 and 41 kDa were also produced in the E. coli minicells. The production of these proteins resulted from additional translational starts within the SdhA coding sequence, suggesting differences between the translational start signals of E. coli and R. prowazekii. Despite the similarity of R. prowazekii SdhA to that of E. coli, the R. prowazekii SdhA did not complement an E. coli sdhA mutant. In addition, analysis of the nt sequence immediately upstream from R. prowazekii sdhA revealed that the rickettsial sdh gene organization differs from that of E. coli and B. subtilis.
Insights
The flavoprotein subunit gene (sdhA) from Rickettsia prowazekii was isolated and sequenced. Despite sequence similarity, R. prowazekii SdhA did not complement an E. coli mutant, indicating functional differences.
Area of Science:
- Molecular Biology
- Bacterial Genetics
- Enzymology
Background:
- Succinate dehydrogenase (SdhA) is crucial for cellular respiration.
- Rickettsia prowazekii is an obligate intracellular parasite with unique metabolic adaptations.
Purpose of the Study:
- To isolate and characterize the gene encoding the flavoprotein subunit (SdhA) of succinate dehydrogenase from Rickettsia prowazekii.
- To investigate the functional and genetic organization of the R. prowazekii sdhA gene.
Main Methods:
- Oligodeoxyribonucleotide probing for gene isolation.
- Nucleotide sequencing and open reading frame (ORF) analysis.
- Heterologous expression in E. coli minicells and SDS-PAGE analysis.
- Functional complementation assays in E. coli sdhA mutants.
Main Results:
- The R. prowazekii sdhA gene was isolated, encoding a 596-amino acid protein with significant homology to other species.
- Heterologous expression revealed multiple translational start sites in R. prowazekii SdhA, differing from E. coli.
- R. prowazekii SdhA failed to complement an E. coli sdhA mutant, suggesting functional divergence.
- The gene organization of rickettsial sdh differs from E. coli and B. subtilis.
Conclusions:
- The R. prowazekii sdhA gene has been successfully isolated and characterized at the molecular level.
- Functional differences exist between R. prowazekii SdhA and its counterparts in other organisms.
- The distinct gene organization highlights evolutionary divergence in Rickettsia metabolism.