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Title of Thesis

Erum Shoeb
Institute/University/Department Details
Department of Genetics/ University of Karachi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
heavy metal tolerance, bacteria, enterobacter sp, pseudomonas aeruginosa, antibiotic tolerance, maximum tolerable concentration, genomic dna, plasmid dna

Isolation of bacteria was done from potentially metal-contaminated areas close to the automobile repair workshops of Karachi, Pakistan. Forty three (43) bacterial strains were isolated which were coded as CMG2K 1-43 and characterized further. Few CMG stock strains were also selected on the basis of their high heavy metal tolerance behavior. These included Enterobacter sp. strain CMG457 and Pseudomonas aeruginosa strain CMG466 which were found to tolerate heavy metals. In case of heavy metals 62.8% of the newly isolated strains showed tolerance against chromate and copper each, and 51.2% against nickel. Isolated strains were also checked for resistance to eight antibiotics: 97.7% of the strains showed tolerance to streptomycin, 44.2% to neomycin, 27.9% to ampicillin, 25.6% to tetracycline, 13.9% to chloramphenicol, 9.3% to kanamycin and novobiocin each, and 2.3% were tolerant to rifampicin. Characterization of all the isolated strains on the basis of different parameters was conducted which include stab culture record, colonial morphology, cellular morphology and their growth curve patterns. Strains were also analyzed for their heavy metal accumulation capabilities and some strains were found to accumulate nickel and copper but none found to accumulate both the heavy metals simultaneously indicating that the mechanisms of accumulation for nickel and copper are different.

Attempt was made to find out correlation between heavy metal tolerance and antibiotic tolerance of these strains with the assumption of multiple stress tolerance of bacteria. Statistical analysis showed positive correlation between heavy metal tolerance and antibiotic tolerance of these strains, t-test at to.05 level of significance has verified positive correlation. Transposon (Ω) Omegon kanamycin containing kanamycin tolerance gene did not hybridize with a Bacillus strain CMG2K8 which has shown tolerance up to 1500 µg/ml of kanamycin, when colony hybridization was performed through DIG labelled probe, this result has signifies that mechanism of tolerance to kanamycin of the strain CMG2K8 is different from the one found in Transposon (Ω) Omegon-kanamycin.

Genetic basis of heavy metal tolerance was analyzed by first screening for the known genes related to heavy metal tolerance within the genome of isolated strains. The genes screened included pco, cop, cueAR operons for copper tolerance and ncc and cnr operons for nickel tolerance. Bacillus cereus strain CMG2K4 and Pseudomonas aeruginosa strain CMG466 that tolerated high concentration of nickel and copper respectively did not indicate presence of any of the related operons Absence of such inducible operons, was confirmed by growth patterns of these strains, which showed similar growth when seeded with overnight cultures grown with and without metal, indicating a constitutive mechanism of tolerance. Most of the heavy metal tolerance mechanisms reported are plasmid borne but in case of these two strains CMG2K4 and CMG466, tolerant against nickel and copper respectively, did not harbor any plasmid confirming that the genes involved in heavy metal tolerance are located on chromosome.

Sequencing of pcoB from CMG457 (Pco operon containing Enterobacter sp. From CMG stock strain) was conducted and same gene was cloned in pGEM-T easy vector and transformed into E. coli JM1 09. SOS-PAGE of crude cell extract of Bacillus cereus strain CMG2K4 in presence of nickel revealed €š36 kDa band which was consistent in log phase culture and stationary phase culture both in control and test, speculated to be the band of a protein involved in nickel tolerance behavior of CMG2K4. N-terminal sequencing yielded 10 amino acid sequence of this €™36 kDa protein showed 100% homology with flagellin protein of several Bacillus cereus strains when MPsrched in Uniprot database of EBI. Multiple sequence alignment has revealed variability in the amino acid sequences of different strains of Bacillus cereus flagellin protein, especially at the C-terminal half of the sequence. MALDI Mass Spectrometry yielded several mass peptide peaks, some of which showed homology in Swissprot database with flagellin protein peptide masses of a number of Bacillus cereus strains.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Content
221.36 KB
2 1 Introduction 7
646.37 KB
  1.1 Heavy Metals 8
  1.2 Occurrence Of Heavy Metals In The Environment 8
  1.3 Consequences Of Heavy Metal Contamination 11
  1.4 Beneficial Heavy Metals 13
  1.5 Toxic Heavy Metals 15
  1.6 Significance Of Microbial Tolerance To Heavy Metals 16
  1.7 History Of Tolerance In Living Cell 18
  1.8 Bacterial Tolerance Against Heavy Metals 18
  1.9 Microbiologist Walk Through Important Heavy Metals 19
  1.10 Influence Of Some Parameters On Levels Of Bacterial Resistance 39
  1.11 Basic Mechanisms Of Tolerance 41
  1.12 Plasmid Mediated Metal Resistance Mechanisms By Bacteria: 43
  1.13 Detailed Studied Organisms For Metal Tolerance 44
  1.14 Protein Families Important For Heavy-Metal Transport 57
  1.15 Biotechnological Use Of Heavy Metal Resistance: An Opinion 63
3 2 Materials And Methods 66
300.95 KB
  2.1 Culture Media And Chemicals 67
  2.2 Selection Of Sampling Sites 67
  2.3 Bacterial Strains And Growth Conditions 67
  2.4 Preservation Of Bacterial Strains 69
  2.5 Revival Of Bacterial Strains 69
  2.6 Characterization of Bacterial Strains 69
  2.7 Growth Curve 70
  2.8 Identification Of Bacterial Strains 71
  2.9 Maximum Tolerable Concentration (MTC) 72
  2.10 Correlation of Heavy Metal Tolerance With Antibiotic Tolerance 73
  2.11 Accumulation of Heavy Metal Salts 73
  2.12 Hybridization 74
  2.13 Isolation Of Genomic DNA 76
  2.14 Isolation of Plasmid DNA 76
  2.15 Designing Of Primers For Polymerase Chain Reactions (PCR) 77
  2.16 Amplification Reaction 77
  2.17 Purification of PCR Product 78
  2.18 DNA Sequencing 78
  2.19 Gel Electrophoresis 78
  2.20 Protein Sequencing 81
  2.21 Multiple sequence alignment 82
  2.22 Peptide Mass Fingerprinting 82
  2.23 Cloning 83
  2.24 Restriction Enzyme Digestion of Genomic DNA 86
4 3 Results 93
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  3.1 Isolation and Purification Of Bacterial Strains 94
  3.2 Selection Of Bacterial Strains From CMG Stock 95
  3.3 Growth Curves 101
  3.4 Identification of Bacterial Strains 104
  3.5 Maximum Tolerable Concentrations 107
  3.6 Statistical Analysis 112
  3.7 Accumulation Of Heavy Metals 114
  3.8 Isolation Of Genomic DNA 116
  3.9 Isolation Of Plasmid DNA 116
  3.10 Dig-Labeled Colony Hybridization 116
  3.11 Genetic Analysis Of Heavy Metal Tolerance 124
  3.12 Restriction Analysis 124
  3.13 Sequencing Of pcob 124
  3.14 Subcloning Of pcob 125
  3.15 Other lanes are showing irrelevant results. Quantitative Analysis Of Protein 132
  3.16 Qualitative Analysis of Protein Through SDS-Page 132
  3.17 Protein Sequencing 132
  3.18 Multiple Sequence Alignment 132
  3.19 Peptide Mass Spectrometry Fingerprinting 133
5 4 Discussion 143
143.41 KB
  4.1 Conclusion 159
6 5 References 161
1074.71 KB