Molecular genetics of arsenic resistance of the biomining bacterium Acidithiobacillus ferrooxidans

Butcher, Bronwyn Gwyneth (2003-12)

Dissertation (PhD)--Stellenbosch University, 2003.

Thesis

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.

AFRIKAANSE OPSOMMING: Acidothiobacillus ferrooxidans, 'n asidofiliese, chemolitotrofiese bakterium, is een van 'n konsortium bakterieë betrokke by biologiese ontgunnig ("biomining") asook by die herwinning van goud uit arsenopiriet erts. Die gene wat aan At. ferrooxidans weerstandbiedendheid teen arseen verleen, is gekloneer. Die DNA-volgorde van hierdie gene is bepaal en daar is bewys dat die gene op die chromosoom geleë is. Homoloë van die arsB (membraan geleë pomp wat arseniet uitpomp), arsC (arsenaat reduktase) en die arsH (funksie onbekend) gene is in bekende arseenweerstanbiedheidsoperons (arsoperons) geïdentifiseer. Verder is daar 'n vierde geen geïdentifiseer wat lae homologie met die ArsR-familie van reguleerders toon. At. ferrooxidans se ars gene is op 'n ongewone manier gerangskik met twee van die gene, arsRC en arsBH wat lil teenoorgestelde rigtings getranskribeer word. Hierdie rangskikking van gene IS waargeneem in al vier die At. ferrooxidans rasse wat getoets is. Al die At. ferrooxidans ars gene is in Escherichia coli uitgedruk. Die arsB en arsC gene het aan 'n E. coli ars mutant (AW311 0) weerstandbiedendheid teen aseniet, antimoniet en arseen verleen. Analiese van die afgeleide aminosuurvolgorde van die ars proteïene het getoon dat die At. ferrooxidans ArsB naby verwant aan die ArsB-proteïene van ander Gram negatiewe bakterieë is. In teenstelling hiermee, is gevind dat die ArsC-proteïene nader verwant aan die ArsC-proteïene van Gram positiewe bakterieë is. Daar is ook gevind dat 'n funksionele tioredoksien (trxA) geen vir ArsC-bemiddelde arsenaat weerstandbiedendheid in E.coli benodig word. Dit dui daarop dat die meganisme van arsenaatreduksie deur At. ferrooxidans soortgelyk is aan die ArsC-proteïen-meganisme van Gram positiewe bakteriee. In vitro studies met behulp van 'n E. coli gebaseerde transkripsie-translasie sisteem het getoon dat arsH nie nodig is vir arsenaat of aseniet weerstanbiedendheid in sensitiewe E.coli rasse nie en ook nie help om weerstand in hierdie rasse te verhoog nie. Daarom kan daar aangeneem word dat die funskie van die arsH geen nie deur E. coli benodig word nie. Die vermeende ArsR van At. ferrooxidans bevat 'n potensiële DNA-binding heliks-draaiheliks motief, maar nie die arsiniet binding motief (ELCVCDL) wat nodig is vir reaksie in die teenwoordigheid van 'n induseerder nie. Die ArsA-proteïen van At. ferrooxidans is soortgelyk aan 'n groep ArsA-proteïene wat tydens genoom DNA- volgordebepalingsprojekte geïdentifiseer is. Hierdie groep gene is egter nog nie verder bestudeer nie. Deur gebruik te maak van 'n stel fusie gene, arsB-IacZ, arsC-IacZ en arsRlacZ kon daar bewys word dat die ongewone ArsH-proteïen van At. ferrooxidans uitdrukking van arsBH en arsRC onderdruk en dat die onderdrukking deur arseniet of arsenaat opgehef kan word. Delesie van die eerste 19 aminosure vanaf die C-terminus van die ArsA-proteïen het geen uitwerking op die regulering van die proteïen nie, maar delesie van 'n vedere 28 aminosure het ArsR geïnaktiveer. Verhoogde vlakke van transkripsie van arsRC en arsBH in die teenwoordigheid van arseniet en arsenaat is met behulp van Noordelike kladanalise bewys. Hierdie is die eerste studie waarin daar bewys word dat die ars gene van die asidofiliese bakterium Atferrooxidans in die neutrofiliese bacterium E. coli bestudeer kan word. Daar is ook bewys dat ten spyte daarvan dat die ArsR in die ars operon nie 'n arseniet bindingsdomein het nie, dit die uitdrukking van die gene in hierdie operon reguleer in reaksie op arseen. Dit dui dus daarop dat hierdie proteïen op arseen in die omgewing reageer met behulp van 'n meganisme wat verskil van die ArsR-proteïene wat tot dusver bestudeer is.

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