Protoplast genetics of a novel halotolerant bacterium (micrococcus sp.).

number: 
304
English
Degree: 
Imprint: 
Biotechnology
Author: 
Bilal Kamil Sulaiman
Supervisor: 
Ghalib H. Al-Bakri
year: 
1998
Abstract:

he effect of different chemical and physical agents on protoplast formation and regeneration of the novel halotolerant bacteria strain Gl, were investigated. The regeneration medium (RM6) of strain Gl was supplemented with different concentrations of NaCl or sucrose or both as osmotic stabilizer preventing the lysis of protoplast. No improvement in the regeneration frequencies were noticed in any treatment compared with the regeneration frequency on the original regeneration medium (RM6) which contain 10% NaCl. Addition of polyethylene glycol or gelatin or glycerol or bovine serum albumin or casamino acids to RM6 medium did not increase the number of regenerants. However gelatin and glycerol enhanced the growth rate of the regenerated colonies but did not increase the number. Some of the physical factors affecting the protoplast formation and regeneration were also tested. Direct spreading method of protoplasts on regeneration medium gave better results than the overlay method. Best regeneration frequencies were obtained when plates were incubated at 35°C. Also fresh RM6 medium was more suitable for regeneration than dry RM6 medium. Cells taken from mid-exponential phase were more suitable for protoplast formation and regeneration than cells taken from late exponential phase or from stationary phase. It was found that concentration of lysozyme f- .used in protoplasting was one of the most important factors in the process of protoplast formation and regeneration. According to the conditions used in this study, treatment of cells with lysozyme with final concentration of 1 mg/ml for 8-12 min. gave the best results. A suitable method for protoplast fusion of strain G 1 was developed during this study. Protoplast fusion frequency was 1.27 x 10"4. This is the first report of successful protoplast fusion system in the halotolerant bacteria. An attempt was made to cure the plasmids of strain G 1 by the protoplast formation regeneration technique. No cured colonies were detected Also a novel transformation system was developed for strain G 1 during this study. Intact cells were successfully transformed with chromosomal DNA. This is the first report of successful transformation system for intact cells of strain G 1 or any other known halophilic and halotolerant bacteria. Several attempt were made to transform the intact cells or the protoplasts of strain Gl and the cured strains GME and GMA with the plasmid cloning vectors pBR322 or pSXl 78 or with plasmid of strain Gl but no transformation was detected in all cases. The protein profile of strain Gl grown on (2%, or 25% NaCl) was investigated and compared with the protein profiles of mutant GM3 (sensitive to 2% NaCl). Mutant GM4 (sensitive to20% NaCl), strain GME (lacking three plasmids and sensitive to 20% NaCl) and strain GMA (lacking two plasmids and sensitive to 20% NaCl).. Results showed the presence of the protein band of about 19000 Dalton in the W.T. strain grown in 2% NaCl but not in the W.T. type strain grown in 25% NaCl. However; a protein band of about 73 000 alton was detected in the strain grown in 25% NaCl but not in the strain grown in 2% NaCl. All the other tested strains showed a protein profile similar to that of the W.T. strain grown in 25% NaCl. It was concluded that the 19000 Daltori protein might play a role in the osmotic regulation of this bacterium when grown in low salt concentration, while the 73000 Dalton protein may be important for the survival of the bacteria in high I salt concentration (10-25% NaCl).