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Title of Thesis

Nazia Jamil
Institute/University/Department Details
University Of Karachi/ Department of Genetics
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
marine bacteria, novel metabolite, pseudomonas aeruginosa, staphylococcus sp., alcaligenes sp, cmg607w, cmg617, cmg634, karachi coast, pha synthese operon

Coastal regions correspond to the interface between terrestrial and marine life. These coastal zones serve as a rich repository of marine organisms among which bacterial flora in these marine environment forms an integral part of this unique ecosystem. To investigate the biodiversity of bacterial flora of Karachi coast, attached and free bacteria were isolated and studied. A total of 36 bacteria (25 attached and 11 free) were isolated from seven different beaches of Karachi, Pakistan (Clifton, Sandspit, Dockyard, Manora, Capmounze, Gadani and Layari out fall). Highest EFUs were observed from sample (Dockyard) using 2216E medium. All the strains were designated as CMG601-CMG635 and one strain was labeled as CMG607w. Among the purified strains most common genera were Bacillus (19%) Escherichia (17%) and Pseudomonas (17%). Most of the strains showed multiple metal and antibiotic resistances. Highest resistance percentage was observed for copper (88%) and ampicillin (86%). Presence of plasmid among free living and attached bacterial were 27% and 36% respectively. Three strains were selected for analysis of metabolite production which were CMG607w; it showed 88% homology to Pseudomonas aeruginosa strain BHP (AY162139), CMG617 showed 90% homology to uncultured Staphylococcus sp. (AF467429) and CMG634 showed 87% homology to Alcaligenes sp (AY346137) for 16S ribosomal RNA gene

When CMG617 was analyzed for metabolite production by non-specific extraction method, four compounds were purified; their molecular formula and mass were calculated. SMHS1 was purified from hexane extract of BHI medium with Rf value 0.6, while its molecular weight was 368.34 amu for C22H44N2O2. From chloroform extract of 2216E medium a compound designated as SMHS3 molecular weight 168.08amu calculated for C8H12N2O2 was purified, while CMG617 also produced fatty acids in the supernatant which were Oleic acid (C17H33COOH; 282.47amu) and plasmatic acid (C15H31COOH; 256.4amu) in presence of 2216E medium

A hygrosopic biopolymer was extracted from CMG34 it absorbed water 4.3 times more than its own weight. Chemically it was found to be glycoprotein. Bioplymer appeard as slime layer and micro fibrils under electron microscope

The third bacterial strain which selected was CMG607w. Metabolite production was analyzed in the supernatant of fermented broths of ASW (supplanted with different carbon sources), it was revealed that in presence of these nutrient conditions CMG607w possess metal binding activity, which may be due to the presence of a compound MW 349 and 41.11kDa protein in the supernatant, detected by GC/MS and phast gel analysis respectively, while EPS production was also observed in presence of different carbon sources

Bioplastic (medium chain length polyhydroxylakanote) was extracted and purified from CMG607w. PHA synthesis was substrate depended in CMG607w. In the presence of 1%sodium gluconate mol-Pha was synthesized at the rate of 42% cell dry mass. In presence of sodium gloconate spherical PHA granules were extruded from the cells, while in presence of sodium acetate, PHA granules were in form of crystals which make the cell fragile and releases from the cell by lyses of the cell membrane. Under highly enriched conditions, co production of polysaccharide and blends of PHB/PHA were observed

PCR based strategy was used to amplify Pha biosynthsis operon from chromosomal DNA. In CMG607w Pha biosynthsis operon has PhaCIZC2D (polymerasel, depolymerase, polymerase2 and hypothetical protein) genes orientation. Conserved sequences were observed in polymerase C1 and C2. All genes of Pha synthase operon were cloned and sequenced. Pha biosynthesis operon of CMG607w has PhaCIZC2D genes and have 98% homology to P. aeruginosa (AE004919, X66592), 87% to P. oleovorans (AF318050) and 77% Burkholderia caryphylli (AF394660) Genbank accession numbers for Pha biosynthesis locus present in CMG607w are ORF1(AY596791), PhaC1((AY5996788), PhaZ (AY596789) PhaC2 (AY5967790) and PhaD (AY596791)

Download Full Thesis
3157.04 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents 0
265.54 KB
2 1 Karachi Coast Quantitative And Qualitative Analysis Of Bacterial Flora 7
315.02 KB
  1.1 Introduction 8
  1.2 Materials And Methods 11
  1.3 Results 16
  1.4 Discussion 32
3 2 Cmg617 (Staphylococcus Saprophyticus ) Analysis For Metabolite Production 36
217.7 KB
  2.1 Introduction 37
  2.2 Materials And Methods 43
  2.3 Results 46
  2.4 Discussion 57
4 3 Cmg634 ( Alcaligenes Sp) Analysis For Metabolite Production 60
517.01 KB
  3.1 Introduction 61
  3.2 Materials And Methods 74
  3.3 Results 77
  3.4 Discussion 89
5 4 Cmg607w Pseudomonas Aeruginosa Analysis For Metabolite Production 92
1062.68 KB
  4.1 Introduction 93
  4.2 Materials And Methods 112
  4.3 Results 120
  4.4 Discussion 144
6 5 Characterization, Cloning And Genetic Analysis Of Pha Synthese Operon In Cmg607w 152
1084.66 KB
  5.1 Introduction 153
  5.2 Materials And Methods 162
  5.3 Results 168
  5.4 Discussion 212
  5.5 Findings 216
  5.6 Literature Cited 218
  5.7 Abbreviations Used In The Manuseript 245
  5.8 Annexure 248