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Title of Thesis

Institute/University/Department Details
University of Karachi/ Department of Genetics
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
pseudomonas aeruginosa cmg103, bacterial strains, dna sequences, heavy metal pollution

Eight bacterial strains, designated as strains CMG101-CMG108, were isolated from metal polluted water samples of the Malir river, Karachi, Pakistan, for their resistance to high concentrations of Zn. Although all strains were identified as Pseudomonas aeruginosa species, they were considered as different strains on the basis of various characteristics. Beside Zn, the CMG strains displayed significant resistance to Cd and some tolerance to Bi and Te. The comparative study of CMG isolates with other known reference strains showed that CMG103 and CMG108 exhibited one of the highest resistance against Zn and Cd reported i.e., up to 8 mM Zn and 2mM Cd

Growth experiments at high temperature and induction experiments indicate that the resistance for Zn and Cd in P.aeruginosa strains CMG103 and CMG108 is linked and controlled by an active metal resistance system. Since both, CMG103 and CMG 108, did not show the presence of plasmids, it was assumed that Zn/Cd resistance is mediated by a chromosomal borne locus. A mutant, czr-13km CMG108#13, impaired in Zn/Cd resistance was obtained in CMG108 by km transposon mutagenesis. Czr-13km was found to be complemented by a genomic 12.8 kb HindIII fragment of CMG103 cloned in pLAFR3. DNA-DNA hybridization studies revealed the presence of czr in a conserved region of P.aeruginosa from different origins

Nucleotide sequence of this fragment demonstrated the presence of four potential genes czrRCBA covering about approximately 6598 bp involved in Zn and Cd resistance and two ORFs which showed similarities with aromatic catabolic genes. The comparison of czrCBA with other sequences in the Genbank EMBL data base demonstrated significant similarities with the structural genes of the plasmid brone metal resistant determinants czr, ncc and cnr. The remarkable similarities with ZcrCBA/CNRCBA which determine a cation-antiporter efflux system suggest that a similar kind of efflux system functions in P.aeruginosa. The predicted protein sequence of the potential regulatory gene, czrR, showed significant similarities cop and pco of P. syringae pv. Tomatoe and E. coli, respwctrively, czrR is located upstream of czrCBA and is divergently described

The heterolgous expression of czr was examined in various strains. In P.fluorescens and A. eutrophus, czr was expressed at low level, while there was no expression in E.coli. However, spontaneous mutants showing increased resistance to Zn and Cd were observed in A.eutrophus due to the insertion of DNA sequences originating from the chromosome of A. eutrophus. These insertion were located in the region containing czrR and the proposed promoter of the czrCBA operon. Insertion of the DNA sequences from A. eutrophus chromosomal DNA in the regulatory region of CZR decreased its expression in P.aeruginosa. This selection was found to be useful to fish out mobile genetic elements in A.eutrophus

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2265.17 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
123.94 KB
2 1 Introduction 3
758.44 KB
  1.1 The Pakistan Environment And Heavy Metal Pollution 4
  1.2 Microbial-Metal Interactions 8
  1.3 Heavy Metal Resistance Determinants In Bacteria 19
  1.4 Pseudomonas Aeruginosa 39
3 2 Materials And Methods 45
260.47 KB
  2.1 Microbiology 46
  2.2 Genetics 50
  2.3 Molecular Biology 52
4 3 Results 64
905.35 KB
  3.1 Selection And Isolation Of Zinc Resistance Bacteria From The Malir River, Pakistan 65
  3.2 Cloning And Characterization And Genetic Analysis Of Zinc And Cadmium Resistance Determinant Of P.Aeruginosa Cmg103 79
  3.3 Expressions Of Czr In Other Bacteria 98
  3.4 Dna Sequence Analysis Of Czr , The Zinc/Cadmium Resistance Determinant Of Pseudomonas Aeruginosa Cmg103 118
5 4 Conclusions, Discussion And Perspectives 171
150.81 KB
  4.1 Czr, A Possible Active Efflux Mechanism For Zn And Cd Resistance In P. Aeruginosa 172
  4.2 Czr, Czc And Their Conservation In Different Bacterial Genera 174
  4.3 Regulatory Aspects 176
  4.4 Adaptation Of The Czr Operon In A. Eutrophus And Is Elements Involved 178
6 5 Litterature Cited 183
336.63 KB