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Title: The development of genetic systems for iron-oxidizing, acidophilic moderate thermophiles
Author: Gibson, F. Elizabeth
ISNI:       0000 0001 3497 4397
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1991
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Acidophilic, moderately thermophilic, Gram positive bacteria which are able to oxidize iron and solubilize sulphide ores are likely to be of industrial importance for the leaching of metals from mineral ores. Genetic manipulation of these bacteria might produce improved strains with regard to desirable leaching characteristics for industry. This work describes initial attempts to develop a host:vector system for the moderate thermophiles. The bacteria were found to be microaerophilic and a pour plate technique and filter disc assay were used to assess the comparative sensitivities of strains ALV, BC1 and TH3 to antibiotics and metals. Chloramphenicol and kanamycin were further investigated as potential selection agents for transformation. The latter was found to be unstable at pH 1.7 and 45°C in a ferrous iron medium. A large plasmid which migrated more slowly than chromosomal DNA during agarose gel electrophoresis was identified in strain 1X2, along with comparatively small plasmids in strains 1X2., IXL, TH1 and BC1. The latter plasmid (pBCl) was cloned into E. coli vectors pACYC177, pBR325 and pMTL20C. These recombinant vectors were used to investigate the host range of pBCl and in vitro expression from the pBCl DNA in an E. coli system. Recombinant vectors containing pBCl did not transform B. subtilis 168 but expressed a polypeptide with apparent M_ of 42,000 as determined by SDS-PAGE following in vitro transcription and translation in an E. coli system. The complete nucleotide sequence of pBCl (2,617 bp) was obtained and encoded four putative open reading frames (ORFs A, B, C and Z) which corresponded to polypeptides of Mp 41,112, 14,227, 8,228, and 6,538 respectively. Analysis of the nucleotide and predicted ORF amino acid sequences Indicated that pBCl belonged to the pC194/pUB110 family of interrelated plasmids from Gram positive bacteria and replicated by a rolling-circle mechanism via a ssDNA intermediate. Evidence for this was the similarity between ORF A of pBCl and other plasmid replication proteins and the identification of a conserved region within the ORF A product (Rep) containing a tyrosine residue which has been shown elsewhere to bind to DNA during replication. Furthermore, a second possible DNA-binding domain was identified within Rep and single- stranded DNA was isolated from strain BC1. A region of the pBCl sequence upstream of Rep was similar to the nick-site within the origin of plus strand replication of pC194 and pUBHO and homology with the minus origins (MO) of these plasmids suggested the presence of an MO in pBCl. A large putative secondary structure of about 100 bases was predicted from the pBCl DNA sequence and was positioned about 250 bases upstream of ORF A and within ORF C. Methodology was developed for the electroporation of strains ALV and BC1 but no electro transformants were isolated. However, a novel method was developed which indicated that plasmid DNA was transferred into the bacteria during electroporation.
Supervisor: Not available Sponsor: Science and Engineering Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology