Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Butcher, Bronwyn Gwyneth"

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    Molecular genetics of arsenic resistance of the biomining bacterium Acidithiobacillus ferrooxidans
    (Stellenbosch : Stellenbosch University, 2003-12) Butcher, Bronwyn Gwyneth; Rawlings, D. E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is one of a consortium of bacteria involved in biornining, including the recovery of gold from arsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans were cloned and sequenced and shown to be chromosomally located. Homologues to the arsB (membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknown function) genes from known arsenic resistance (ars) operons were identified. A fourth gene was found to have weak homology to the ArsR-family of regulators. The arsenic resistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRC and arsBH genes divergently transcribed. This divergent arrangement was found to be conserved in all four of the At. ferrooxidans strains we tested. All of the At. ferrooxidans ars genes were expressed in Escherichia coli and the arsB and arsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to an E. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these ars genes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteins from other Gram-negative bacteria. However, the ArsC protein is more closely related to the ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin (trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggests that reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that by Gram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitro transcription-translation system, the presence of this gene was not required for, nor enhanced, arsenite or arsenate resistance in E. coli. We predict that the function provided by this gene is not required in E. coli. While the putative ArsR from At. ferrooxidans does contain a potential DNA-binding helix-turn-helix (HTH) domain, it does not contain the arsenite binding motif (ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-like protein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been associated with other ars-like genes identified during genome sequencing projects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that this atypical ArsR protein from At. ferrooxidans did repress expression from the arsBH and arsRC promoters and that this repression was relieved by the presence of either arsenite or arsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did not affect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northern blot hybridization confirmed that expression of the arsRC and arsBH transcripts is increased in the presence of either arsenite or arsenate. This study is the first to show that the ars genes from the acidophilic biorning bacterium At. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We have also shown that the atypical ArsR found in this ars operon is able to regulate expression of these genes in response to arsenic, despite not containing the arsenite binding domain, suggesting that this protein senses arsenic by a different mechanism to that used by the ArsR family members already studied.