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Title: The regulation of arsenic metabolism in Rhizobium sp.str. NT-26
Author: Corsini Madeira, P.
ISNI:       0000 0004 8498 9580
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Arsenic (As) is a toxic metalloid and a major contaminant in terrestrial and aquatic environments. The two soluble forms, arsenite (AsIII) and arsenate (AsV) are toxic to most organisms. A range of phylogenetically distant bacteria are able to oxidize AsIII to the less toxic form, arsenate AsV using the periplasmic arsenite oxidase (AioBA). The two-component signal transduction system AioS/AioR and the AsIII-binding periplasmic protein AioX are required for AsIII oxidation and are involved in the transcriptional regulation of the aioBA operon. Most AsIII oxidisers can also reduce AsV to AsIII via the As (Ars) resistance system. The focus of this work was to understand the regulation of genes involved in AsIII oxidation and As resistance together with those involved in phosphate metabolism in the facultative chemolithoautotrophic AsIII oxidiser NT-26 grown under different conditions. Gene expression was studied by quantitative PCR in cells grown heterotrophically with and without AsIII or AsV in late-log and stationary phases. qPCR was optimised and suitable reference genes were chosen. The expression of genes involved in phosphate transport, sensing As and the genes aioX, aioS, aioR (AsIII-sensing and regulation) and aioB, aioA (AsIII oxidation) and cytC (cytochrome c) were also analysed in NT-26 grown heterotrophically in the presence or absence of AsIII or AsV at different growth stages (i.e., late-log and stationary phases). To understand gene expression in more detail the transcription start sites (TSS) upstream of aioX, aioB and aioA were identified using the 5'RACE technique. Promoter function was then confirmed by cloning the putative promoter regions upstream of a promoterless β-galactosidase gene and enzyme activity measured in NT-26 grown heterotrophically with and without AsIII or AsV at different growth stages. Two functional promoters were found to control the expression of the aioXSR operon, one for its constitutive expression and another for expression in stationary phase. Surprisingly a TSS was identified upstream of aioA and the presence of a functional promoter was validated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available