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Title of Thesis

Riaz ul Haq
Institute/University/Department Details
Department of Zoology, University of the Punjab, Lahore
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
GENOME ORGANIZATION, THIOBACILLUS FERROOXIDANS, DNA sequences, T. acidophilus, T. thioparus, T. novellus, Leptospirillum ferrooxidans, Acidophiliun, cyrptum, A organovorum, Escherichia coli

Twenty-two strains of Thiobacillus ferrooxidans, obtained from various localities of the world, were analyzed for the presence of family 1 and family 2 repeated DNA sequences by the Southern not technique. Evidence shows that these sequences transpose in the genome of T. ferrooxidans and help the bacterium adapt to changing environment of various salt concentrations and temperatures. Twelve strains, including the type strain ATCC 2370 have multiple copies of both families. One strain has family 1but not family 2, three strains have family 2 but not family 1 and the remaining six strains have neither of the families. Three strains of Thiobacillus thiooxidans were similarly analyzed. Two of the strains have both families of repeated DNA sequences and the other strain have neither family.

Analysis of restriction fragment length polymorphism indicated that the multiple copies of both, family 2 are conserved within and between strain of T. ferrooxidans and also between T. thiooxidans. In contrast the genomic locations in which members of family 1 and family 2 are found can vary between strains of T. ferrooxidans. An analysis of these positional differences suggests that both families are insertion sequence elements capable of transposition.

The following bacterial strains lacked family 1 and family 2 repeated sequence T. acidophilus, T. thioparus, T. novellus, Leptospirillum ferrooxidans, Acidophiliun, cyrptum, A organovorum and Escherichia coli C600

Four Thiobacillus ferrooxidans strain isolated from effluents of coal-mines of Pakistan were screened for the presence of plasmids. One of the strains designated L9 (from Lakhra coal-mines of Sind Province ), was found to have plasmid. The purity of the strain was assessed by DNA probes through Southern blotting. Plasmid DNA from T. ferrooxidans L9 was isolated and used to transform E. coli HB101. Transformants were selected on ampicillin plates. Plasmid DNA from transformant E. coli cells was isolated and the size of DNA was compared with plasmid DNA of T. ferrooxidans L9. The transformant E. coli HB101 cells were grown on plates containing different concentration of 9 antibiotics and at least three metal ions. In this study at least eig selectable markers were noted on a T. ferrooxidans plasmid.

A pure culture of T. ferrooxidans growing in basal salts- thiosulfate medium was used to determine growth of T. ferrooxidans on organically supplemented thiosulfate agarose plates. Fourteen organic compounds , i.e. amino acids vitamins were us in a concentration of 10 µg/ml in plates. T. ferrooxidans showed growth on 12 organ compounds, while no growth was observed on two of the compounds. The study would open a way to determine growth conditions of T. ferrooxidans mutagenesis a selection of auxotrophs. Cultures of T. ferrooxidans growing on ferrous sulfate thiosulfate and tetrathionate media transferred from one mediums to other a their ability to switch from one energy source to other was observed. T. ferrooxidans showed ability to switch from one source to other in some cases while in other cases showed inability to grow on changed medium, in this study.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
178.25 KB
2 1 Introduction 1
415.99 KB
  1.1 Thiobacillus Ferrooxidans As The Key Bacterium In Metal Recovery Operation 5
  1.2 Genome Of T. Ferrooxidans On Different Media 7
  1.3 Genome Organization Of T. Ferrooxidans 8
  1.4 Plasmid Isolation From T. Ferrooxidans 8
  1.5 Aims And Objectives Of The Study 8
3 2 Materials And Methods 12
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  2.1 Bacterial Strains 12
  2.2 Media 14
  2.3 9k Ferrous Media 14
  2.4 Thioslufare Medium 14
  2.5 Tetrathionare Medium 15
  2.6 Dna Preparation 16
  2.7 Detection O Plasmid 17
  2.8 Isolation And Purification Of The Plasmid 17
  2.9 Agarose Gel Electrophoresis 18
  2.10 Transformation 18
  2.11 Antibiotics And Metal Ion Containing Plates 19
  2.12 Growth And Change Of Media 20
  2.13 Organic Supplements Media 20
  2.14 Nick Translation 20
  2.15 Southern Blotting 21
  2.16 Preparation Of Probes 24
4 3 Results 24
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  3.1 Construction Of Dna Probes 24
  3.2 Presences Of Family 1&2 Repetitive Dna In Strain Of T. Ferrooxidans And T. Thiooxidans 29
  3.3 Analysis Of T. Ferrooxidans Strains Strain With Signal Copy Probes 36
  3.4 Conservation Of Family 1 And Family 2 Repetitive Dna Sequence 37
  3.5 Positional Analysis Of Family 1 And Family 2 38
  3.6 Detection Of Plasmid In T. Ferrooxidans Strain 35
  3.7 Resistance Of The Transformant Against Antibiotics 45
  3.8 Resistance To Metal Ions 45
  3.9 Growth Of T. Ferrooxidans On Organically Supplemented Media 50
5 4 References 65
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