Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient

Abstract The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.