Molecular genetics of arsenic resistance of the biomining bacterium Acidithiobacillus ferrooxidans

[摘要] ENGLISH ABSTRACT:The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is oneof a consortium of bacteria involved in biornining, including the recovery of gold fromarsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans werecloned and sequenced and shown to be chromosomally located. Homologues to the arsB(membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknownfunction) genes from known arsenic resistance (ars) operons were identified. A fourthgene was found to have weak homology to the ArsR-family of regulators. The arsenicresistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRCand arsBH genes divergently transcribed. This divergent arrangement was found to beconserved 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 andarsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to anE. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these arsgenes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteinsfrom other Gram-negative bacteria. However, the ArsC protein is more closely related tothe ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin(trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggeststhat reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that byGram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitrotranscription-translation system, the presence of this gene was not required for, norenhanced, arsenite or arsenate resistance in E. coli. We predict that the function providedby this gene is not required in E. coli.While the putative ArsR from At. ferrooxidans does contain a potential DNA-bindinghelix-turn-helix (HTH) domain, it does not contain the arsenite binding motif(ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-likeprotein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins thathave been associated with other ars-like genes identified during genome sequencingprojects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that thisatypical ArsR protein from At. ferrooxidans did repress expression from the arsBH andarsRC promoters and that this repression was relieved by the presence of either arsenite orarsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did notaffect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northernblot hybridization confirmed that expression of the arsRC and arsBH transcripts isincreased in the presence of either arsenite or arsenate.This study is the first to show that the ars genes from the acidophilic biorning bacteriumAt. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We havealso shown that the atypical ArsR found in this ars operon is able to regulate expressionof 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 theArsR family members already studied.

[发布机构] Stellenbosch University