Temperature-Sensitive Mutants of Escherichia coli Affecting β-Galactoside Transport

Abstract

Six different temperature-sensitive ( ts ) mutants have been isolated which have parental β-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from Escherichia coli ML308 ( lacI − Y + Z + A + ). After N -methyl- N ′-nitro- N ′-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-β- d -galactoside uptake and in vivo o -nitrophenyl-β- d -galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile lacY gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a ts increase in permeability as shown by the increased leakage of intracellular β-galactosidase and by the increased rate of in vivo o -nitrophenyl-β- d -galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have ts alterations in membrane synthesis which results in pleiotropic effects on various membrane functions.