On the molecular biology of a novel halotolerant bacterium (micrococcus sp.)

number: 
222
English
Degree: 
Imprint: 
Biotechnology
Author: 
khalaf J. Al-Dolaimi
Supervisor: 
Ghalib H. Al-Bakri
Dr. Jaladet M. S. Jubra'el
year: 
1997
Abstract:

Ultra Violet survival curves of the novel halotolerant bacteria strain Gl (Mjcrococcus sp) Escherichia coli & Staphylococcus aureus showed that strain Gl was much more resistant to U.V radiation than E coli & S_. aureus. Types of DNA repair systems present in this novel halotolerant bacterium was investigated. Strain Gl was irradiated with different doses of U.V. light, part of irradiated cells were exposed to sunlight after irradiation, while the other kept in the dark. It was found that the survival fraction of the cells exposed to light was higher than that of the cells kept in the dark, indicating that strain Gl possesses a photoreactivation system. Strain Gl cells were also U.V. irradiated & plated on YMC agar alone ft on YMC agar containing different sub'ethal concentrations of either caffeine (0, 2000, 3000, A 4000 ug/ml) or acriflavine (0, 0.2, 0.4 A 0.6 ug/mI). The survival of strain Gl in the presence of caffeine & acriflavine was much lower compared with that on YMC agar alone indicating that this bacterium possesses excision repair & recombination repair, in addition to the photoreactivation repair. Total genomic DNA of strain Gl was isolated, purified & subjected to restriction analysis with six restriction enzymes recognizing hexanucleotide sequences ( Eco RI, Hin dIII, Bam HI, Pst I, Sal I & Sma I). Some of these enzymes recognize a GC rich sequences, other recognize an AT rich sequences. Genomic DNA of strain Gl was generally more sensitive to the enzymes recognizing GC rich sequences indicating that this bacterium has a hiph GC content. An attempt was made to construct a genomic library of this strain using the plasmid pBR322 as a cloning vector. Two methods were pursued for this purpose: the first was by inserting Sau 3 A partially digested genomic DNA of strain Gl into the Bam HI site of pBR322, the second was by inserting Hin dIII completely digested genomic DNA of Gl into the Hin dIII site of pBR322. Conditions for partial digestion with Sau 3 A were optimized. Plasmid pBR322 was isolated from E coli HB101 A linearized with either Bam HI or Hin dIII.Sau 3 A partially digested DNA was ligated into the Bam HI site of pBR322 & completely digested genomic DNA with Hin dIII was ligated into the Hin dIII site of the same plasmid. Ligation mixtures & intact pBR322 as a control, were used to transform E coli HB101 & selecting for ApR transformants. No transformants were detected from the ligated mixtures, while large No. of transformants were obtained from using intact pBR322 (transformation ligation-frequency 8x 10"5) . Ligated mixures were run on agarose gel to detect