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dc.contributor.authorImran, Maimoona-
dc.description.abstractSilver is a non-essential transition metal that becomes hazardous at higher concentrations. Because of industrialization, its concentration has increased in soil, water and biological systems. Many organisms including bacteria have developed resistance against this metal. In the present study, silver resistance potential and mechanism involved have been investigated in a Klebsiella pneumoniae strain KW locally isolated from effluent collected from Kot Lakhpat industrial zone. K. pneumoniae KW was able to grow in the presence of high concentrations of silver. Minimum inhibitory concentration of silver for this bacterium was found to be 80mM. Effect of silver on growth of K. pneumoniae KW was studied by comparing its growth in the presence of various concentrations of silver in the medium with that in the absence of the metal. Addition of silver in early log phase culture initially allowed culture to grow (for 2 hs) followed by halted growth. Further detectable increase in growth was observed in 24 hs old culture. K. pneumoniae KW exhibited remarkable silver uptake ability. In the presence of 20, 30 and 60mM Ag+ in the medium, 104.92, 275.5 and 390.2 μg Ag+ was found uptaken by each mg cell dry weight, respectively. This study first time reports role of Cus and Cue determinants for silver resistance in K. pneumoniae. Transcripts level of cusRS and cus CFBA (cus regulon) and copA, cueO and cueR (cue regulon) were determined in the presence of 0, 1, 2, 3, 4 and 5 mM silver after 15 min of metal stress. Presence of silver led to significantly enhanced transcription of all the genetic determinants studied. CusRS constitutes a two component regulatory system. It comprises of a histidine kinase CusS and a response regulator CusR. Both cusR and cusS were amplified from genomic DNA of K. pneumoniae KW, cloned in pTZ57R cloning vector and subjected to nucleotide sequencing. cusR and cusS was sub-cloned in pET-21a and pET-28a expression vectors, respectively. Recombinant CusR and CusS were expressed in BL21 codon plus competent cells, transformed with respective recombinant vectors. Expression analysis revealed that maximum expression of CusR was obtained in the presence of 0.1mM IPTG with 4h induction. For purification of CusR, total cell proteins of BL21 cells transformed with recombinant pET-21a/cusR vector were subjected to ammonium sulphate precipitation followed by FPLC. Purified CusR protein was characterized by determining its binding ability with bi-directional cus promoter. For this, CusR protein 9 was incubated with amplified and purified pcus promoter. Gel shift assay results showed that CusR protein had great binding affinity with the promoter. An effort was made to crystalize CusR protein. CusR was sub-cloned in pET22b expression vector, expressed in BL21 codon plus cells, and purified by immobilized affinity chromatography followed by size exclusion chromatography. However, protein got precipitated and thus could not be subjected to crystallization. Construction of homology-based three-dimensional models and analyses through Conserved Domain Database and UniProt revealed the presence of conserved domains in both regulatory CusR and CusS proteins. Protscale analysis of these proteins gave an idea about the physical and chemical properties; accessible and buried residues, hydrophobicity of certain regions, mutability, polarity and the amino acids responsible for these properties. String database analysis of CusR has shown its interaction with RspR, CutA, ZraS and CueO exhibiting its role in cellular response to silver copper and zinc ions, protein homo-oligomerization, signal transduction, peptidyl histidine phosphorylation, detoxification and transport of ions across the membrane and biological regulation. A multiple node interaction of CusS was also determined that showed close interactions of CusS with CutA, CueO, YedW, CitA and Dcus. It also showed the functions of CusS other than sensing copper and silver; involvement in detoxification of metal ions, signal transduction, protein modification and ion export across the membrane. This endeavor demonstrates that the local isolate Klebsiella pneumoniae KW is able to survive in the presence of high concentrations of silver and possess the intrinsic ability to uptake silver. At molecular level, silver regulates the expression of cus determinants that encodes for proteins involved in silver sensing, activation of the cus system and efflux of this metal.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.publisherUniversity of the Punjab, Lahoreen_US
dc.subjectNatural Sciencesen_US
dc.titleMolecular Characterization of Cus Regulon in Response to Silver in Klebsiella Pneumoniaeen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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