Mutant of Escherichia coli K-12 deficient for detergent-resistant phospholipase A.
A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.
D002247:Carbon Isotopes; D002474:Cell-Free System; D002855:Chromatography, Thin Layer; D002999:Clone Cells; D003902:Detergents / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D005227:Fatty Acids / Q000096:biosynthesis; D005827:Genetics, Microbial; D006868:Hydrolysis; D008242:Lysogeny; D009153:Mutagens; D009154:Mutation; D009604:Nitrosoguanidines; D010740:Phospholipases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
D002247:Carbon Isotopes; D003902:Detergents; D005227:Fatty Acids; D009153:Mutagens; D009604:Nitrosoguanidines; D010743:Phospholipids; D012967:Sodium Dodecyl Sulfate; D010740:Phospholipases