Abstract
We have developed a rapid autoradiographic colony assay for detecting mutants with elevated levels of certain biosynthetic enzymes. Four Escherichia coli strains in which the specific activity of the membrane enzyme diglyceride kinase is increased 5-10-fold have been obtained with this approach. The mutant kinase has the same thermal denaturation profile and subcellular localization as the wild type. Five other membrane enzymes involved in phospholipid bilayer assembly are unaffected. In one of these strains (GK-1) the mutation (dgkR-1) responsible for the elevated kinase has been mapped at a new site near minute 92, while the previously identified structural gene (dgk) lies near minute 90. When the structural gene for the kinase (dgk) is cloned on a multi-copy vector-like ColE1, the kinase can be overproduced 5-10-fold on the basis of gene dosage (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420). Introduction of such hybrid plasmids into a mutant harboring dgkR-1 leads to a multiplicative (rather than additive) effect, resulting in specific activities of diglyceride kinase that are 35-75-fold higher than normal. These results show that dgkR-1 is a trans-acting mutation and suggest the existence of novel regulatory proteins (or metabolites) that direct the expression of certain membrane enzymes.
Highlights
Tually regulate the expression of these genes are unknown
Madison, Wisconsin 53706 promoters exist in this system, we have developed a general strategy for detectingE . coli mutants with elevated levels of
We have developed a rapid autoradiographic colony biosynthetic enzymes
Summary
Madison, Wisconsin 53706 promoters exist in this system, we have developed a general strategy for detectingE . coli mutants with elevated levels of. We have used forthis purpose the rapid assay for detecting mutants with elevated levels of in situ colony autoradiography proceduredescribed earlier for certain biosynthetic enzymes.FourEscherichia coli obtaining enzymatically defective strains (10, l l ) , except that strains in which the specificactivity of the membrane thepresentstrategy employs shorttermassaystolocate enzyme diglyceride kinase is increased 5-10-fold have colonies with greater than normal syntheticcapacity. The identification of strains harboring synthesis of these membrane proteins appears to be consti- hybrid ColEl plasmids bearing the dgk structural gene (Table I) has tutive undera variety of culture conditions [1].A few of them have been purified to homogeneity [1,2,3], and each represents about 0.01 to 0.1% of the totalcellular protein, approximately equivalent to 1000 polypeptide chains/enzyme/cell. Protein was measured by the method ofLowry et al [17], while radioactive lipid preparations were analyzed by means of liquid scintillation spectrometry, employing 10 ml of Patterson-Greene fluid as the scintillation mixture [18]
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