Effect of physical and chemical mutagens staphylococcus aureus cell wall.+CD

This study aimed to modify Staphylococcus aureus by using physical andhemical mutagens and subjections the cell wall to the hydrolytic activity of lysozyme. Chemical mutagenesis by N-methyle-N-nitro-N-nitroguanidine, was achieved by incubating the cell suspension of Staphylococcus aureus with 100μg/ml MNNG for different periods of time (5, 10, 15, 20, 25 and 30min). Aliquot of 100μl of the bacterial suspension subjected to the lethal and mutagenic effect of MNNG, then spread on BHI agar and incubated at 37°C over night in dark. After incubation, 180 colonies were randomly selected and screened for their ability to grow on the same medium supplemented with 3.4 and 12.5μg/ml lysozyme to detect the lysozyme sensitive mutants. Results showed that all of the selected colonies were able to grow on 12.5μg/ml lysozyme containing medium except one colony (S1). Physical mutagenesis by UV radiation was achieved by subjecting cell suspension of S. aureus to different doses of UV radiation (1, 2, 3, 4 and 5J/m2). Aliquot of 100μl of the bacterial suspension was exposed to the lethal and mutagenic effect of UV radiation then spread on BHI agar and incubated at 37°C over night in dark. After incubation, 160 colonies were randomly selected and screened for their ability to grow on the same medium but containing 3.4 and 12.5μg/ml lysozyme. Results showed that all of the selected colonies were able to grow on these media except four colonies (S2, S3, S4, and S5) which were unable to grow on 12.5μg/ml lysozyme containing medium, these four colonies were considered to be lysozyme sensitive mutants. Plasmid profile of S. aureus was studied; the wild type and the lysozyme sensitive mutants (S1, S2, S3, S4, and S5) using alkaline lysis method, the total genomic DNA was extracted by lysozyme instead of lysostaphin which is known for its ability to lyse S. aureus, results showed the appearance of chromosomal band in addition to plasmid band from all of the lysozyme sensitive mutants (S1, S2, S3, S4, and S5), however the method was unable to extract DNA from the wild type because S. aureus cell wall is resistant to the hydrolytic effect of lysozyme, this result confirmed that chemical and physical mutagens can cause alternation in the structural genes responsible for the synthesis of the cell wall. The synergism effect of penicillin and lysozyme on S. aureus cell wall was studied. Results showed that incubation of S. aureus with the minimal inhibition
concentration MIC of penicillin (640u/ml) for different time periods (2, 3, 4 and 5 hours) then incubating the cells with 250μg/ml lysozyme for two hours causes the gradual lysis of the cell wall and finally the complete lysis as the incubation time progress by the formation of protoplast. Genomic DNA was extracted from these cells by alkaline lysis method using lysozyme instead of lysostaphin; results showed the appearance of chromosomal band and plasmid band on 0.7% agarose gel. Lysozyme sensitive mutants isolated by UV and MNNG mutagenesis were characterized and the results were compared with the wild type by studying antibiotic sensitivity pattern for the wild type and for the five mutants resulted after mutagenesis with chemical and physical mutagens. Results showed variations in the antibiotic sensitivity pattern against the twelve antibiotics that were used in comparison with the wild type; these variations could be a result of the mutation in the genes responsible for the antibiotic resistance caused by the physical and chemical mutagenesis. The possibility of lysing the cell wall of S. aureus mutant cells by lysozyme (50μg/ml) alone to the cell suspension results of observing the microscopic changes showed that S. aureus was converted into protoplasts after incubation for 2hours. This result confirms that the hydrolytic activity of lysozyme against mutant's cells of S. aureus can be used to acquire protoplast unlike the wild type in which the lysis of its cell wall requires a long period of incubation with penicillin and higher concentration of lysozyme. To identify the changes in the growth conditions of S. aureus after the chemical and physical mutagenesis in comparison with S. aureus wild type, mutant cells were incubated at different temperature (30, 37, 40, 43, and 45°C). Results showed that unlike the wild type some of the mutants were unable to grow at high temperature (over 40°C), suggesting defects in cell surface structure and the membrane integrity caused by the mutagens effect.