Mutants of escherichia coli with abnormal patterns of repression of arginine biosynthetic enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
Experimental work was done to attempt to isolate further mutants of the arginine regulatory gene in Escherichia coli W of the same type as a strain known as W2-250, This mutant carries the argR*
regulatory gene which confers upon it the diminished ability to repress or derepress its arginine biosynthetic enzymes. Experimental work involved establishing a suitable method of exaggerating the slow growth rate of this strain on the arginine intermediate N -acetyl-L-ornithine, such that this could be the basis of protecting similar mutants when selecting them from a parent strain (W2-40) in a penicillin select-ion procedure. This was achieved by growing both strains in diphasic a medium containing 15ug/cm3
of L-arginine and an excess (30ug/cm3
) of acetylornithine, in which it was found that the argR strain entered & premature stationary phase when the arginine was exhausted but the argR strain continued growth at a slower rate than normal in the acetylornithine. It was also found that a culture of W2-250 which had entered this stationary phase could be diluted 1:1 in fresh minimal medium + acetylornithine and still remain in a stationary condition for up to 24 hours. Mutants of strain W2-40 produced using ultraviolet light were cycled twice through a system involving this stationary phase in diphasic medium and dilution 1:1 in fresh medium + 500 units of benzyl-penicillin/cm3
in order to select against the parent-type. Further selection was carried out on minimal agar plates supplemented with acetylornithine and canavanine on which the parent strain will grow, but the argR* type is inhibited until arginine was added to the agar. The colonies which appeared at this stage were screened on variously supplemented solid media to select those most like W2-250. Those selected on these criteria were then screened using the acetylornithinase assay and the diphasic medium, with W2-250 included for comparison. A total of nine possible mutants of the desired type were isolated, but problems with the preparation of enzyme samples precluded a definite decision on their identity. In the course of this work it was also discovered that an arg8
bradytroph (W2-25/8) could behave similarly to W2-250 in diphasic media. It was also found that there was some transferable "factor" in the medium of cells which had reached the premature stationary phase which could prevent further growth of either W2-250 or W2-25/8 in non-repressive minimal medium. This is discussed in detail.