The study of two genes involved in the maintenance of intracellular redox potentials in Thiobacillus ferrooxidans
Doctoral Thesis
1996
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University of Cape Town
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The thioredoxin and ϒ-glutamylcysteine synthetase genes of Thiobacillus ferrooxidans were isolated by complementation of an Escherichia coli trxA gshA double mutant for growth on minimal medium lacking glutathione. Transduction of a T. ferrooxidans genome cos mid library into the E. coli mutant resulted in the identification of two groups of complementing cosmids. One of these groups was able to complement the TrxA⁺ requirement of an E. coli trxA met mutant for conversion of methionine sulfoxide to methionine. The nucleotide sequence of a 1.1 kbp Hindiii-Psti T.ferrooxidans chromosomal DNA fragment containing the cloned trxA gene, was determined. An open reading frame (ORF) corresponding to the thioredoxin gene and part of an ORF corresponding to the N-terminal region of the rho gene were identified. A 14 kDa protein corresponding to the T. ferrooxidan thioredoxin was synthesised in an E. coli-derived in vitro transcription/translation system. The predicted amino acid sequence of the T.ferrooxidans trxA gene showed 70.6% identity to that of E. coli. The cloned T. ferrooxidans trxA gene was able to support T7 phage replication in an E. coli trxA mutant to almost the same level as a TrxA⁺ wild type strain. However, it was not able to satisfy the thioredoxin requirement of M13 phage. Crude extracts of the E. coli trxA gshA mutant containing the cloned T. ferrooxidans trxA gene were able to reduce insulin far more rapidly those that did not contain the cloned gene, indicating that the T.ferrooxidans thioredoxin has disulfide reductase activity. DNA:RNA hybridisation analysis was carried out on transcripts prepared from the E. coli trxA gshA double mutant, the mutant containing the cloned trxA gene and T. ferrooxidans cells. A single transcript of about 0.5 kbp was obtained for RNA from T. ferrooxidans cells. Several transcripts were produced from the cloned T. ferrooxidans trxA gene in E. coli. One of these transcripts corresponded in size to the 0.5 kbp transcript produced by T. ferrooxidans cells, whereas the other transcripts were larger (1.35, 1.40, 1.60 and 1.80 kbp) and presumably represented transcription products originating from the lac promoter of the vector. Primer extension analysis of the thioredoxin gene with RNA prepared from T. ferrooxidans gave three transcription start sites. RNA derived from the cloned trxA gene in E. coli only gave two transcription start sites, which were identical to two of the three found with T. ferrooxidans RNA. The second group of cosmids which was able to complement the gshA trxA double mutant for growth on minimal media but which did not allow growth of the E. coli trxA met mutant on minimal media plus methionine sulfoxide was also examined. The nucleotide sequence of a 2.3 kbp Clai-BamHI T. ferrooxidans chromosomal DNA fragment containing the complementing DNA, was determined. Two ORFs, separated by 9 bp, corresponding to a citrate synthase gene and a ϒ-glutamylcysteine synthetase gene were identified. The predicted amino acid sequence of the T. ferrooxidans gltA gene showed 37% identity to that of E. coli, whereas the predicted amino acid sequence of the T. ferrooxidans gshA gene only showed 18% identity to that of E. coli. The low homology of the gshA gene products is not abnormal, as the ϒ-glutamylcysteine synthetases studied to date differ widely in amino acid sequence and structure. Partial sequencing of the cloned T. ferrooxidans chromosomal DNA upstream of the gltA gene indicated regions of homology to the genes of the pyruvate dehydrogenase complex. Synthesis of proteins corresponding to the T. ferrooxidans citrate synthase (43 kDa) and ϒ-glutamylcysteine synthetase (49 kDa) enzymes were confirmed using an E. coli-derived in vitro transcription/translation system. The identity of the proposed citrate synthase gene was confirmed by complementation of an E. coli gltA mutant. Similarly, the DNA upstream of the gltA gene, was found to complement an E. coli mutant for the pyruvate dehydrogenase complex, confirming that this region contained the T. ferrooxidans pyruvate dehydrogenase complex, as predicted from the partial sequence analysis. The identity of the T. ferrooxidans ϒ-glutamylcysteine synthetase gene was confirmed by determining the levels of glutathione in crude extracts of the E. coli gshA trxA double mutant, the mutant containing the cloned T. ferrooxidans gshA gene, and an E. coli parental strain. Crude extracts prepared from E. coli double mutant contained much lower levels of glutathione than crude extracts prepared from the E.coli mutant containing the cloned gshA gene, indicating that the gene from T. ferrooxidans encoded a protein with ϒ-glutamylcysteine synthetase activity. RNA:DNA hybridisation analysis was carried out using probes specific to the gshA gene, the gltA gene and the aceF gene (pyruvate dehydrogenase transacetylase gene). All three probes gave transcripts of 9 kbp and a number of smaller transcripts with RNA derived from T. ferrooxidans cells. The 9 kbp transcripts indicated transcriptional linkage of the y- glutamylcysteine synthetase gene, the citrate synthase gene and the genes of the pyruvate dehydrogenase complex in T.ferrooxidans.
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Bibliography: p. 128-155.
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Reference:
Powles, R. 1996. The study of two genes involved in the maintenance of intracellular redox potentials in Thiobacillus ferrooxidans. University of Cape Town.