ISOLATION OF LACTOSE PERMEASE MUTANTS WHICH RECOGNIZE ARABINOSE

In the present study lactose permease mutants were isolated which reco gnize the monosaccharide, L-arabinose. Although the wild type permease exhibits a poor recognition for L-arabinose, seven independent mutant s were identified by their ability to grow on L-arabinose minimal plat es. When subjected to DNA sequencing, it was found that all seven of t hese mutants were single-site mutations in which alanine 177 was chang ed to valine. The wild type and valine 177 mutant were then analyzed w ith regard to their abilities to recognize and transport monosaccharid es and disaccharides. Free L-arabinose was shown to competitively inhi bit [C-14]-lactose transport yielding a K(i) value of 121 mM for the V al177 mutant and a much higher value of 320 mM for the wild-type. Amon g several monosaccharides, D-glucose as well as L-arabinose inhibited lactose transport in the Val177 mutant to a significantly greater exte nt, while D-arabinose and D-xylose only caused a slight inhibition. On the other hand, kinetic studies with sugars which are normally recogn ized by the wild-type permease such as [C-14]-galactose and [C-14]-lac tose revealed that the Val177 mutant and wild-type strains had similar transport characteristics for these two sugars. Overall, these result s are consistent with the notion that the Val177 substitution causes a n enhanced recognition for particular sugars (i. e. L-arabinose) but d oes not universally affect the recognition and unidirectional transpor t for all sugars. This idea is further supported by the observation th at site-directed mutants containing isoleucine, leucine, phenylalanine , or proline at position 177 also were found to possess an enhanced re cognition for L-arabinose.