FUNCTIONAL ROLES OF GLU-269 AND GLU-325 WITHIN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Acidic residues which are found on transmembrane segments within the l actose permease may play an important role in H+ and/or sugar recognit ion. To examine the functional roles of Glu-269 and Glu-325, we have c onstructed a variety of amino acid substitutions (e.g. aspartate, glyc ine, alanine, serine, or glutamine) via site-directed mutagenesis. At position 269, all mutations appear to have a detrimental effect on sug ar affinity, downhill transport, and counterflow. The Asp-269 mutant w as able to accumulate lactose against a concentration gradient, wherea s all of the nonionizable substitutions at position 269 were completel y defective. Nevertheless, in spite of their inability to actively acc umulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides. Mutations at position 325 had a markedly different phenotype. For example, the Asp-325, Gly -325, and Gln-325 mutants exhibited an apparent K-m for lactose transp ort (e.g. 0.21, 0.47, and 0.50 mM, respectively), which was actually l ower than that of the wild-type strain (1.44 mM). In counterflow assay s, all position 325 mutants also appear to catalyze lactose exchange. Similar to the results obtained at position 269, the Asp-325 mutant ex hibited moderate levels of accumulation, whereas none of the nonioniza ble mutations at position 325 were able to accumulate galactosides aga inst a concentration gradient. However, unlike the position 269 mutant s, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or G ln-325 strains upon the addition of lactose, -beta-D-galactopyranosyl- (1,1)-beta-thiogalactopy- ranoside, 1-O-methyl-beta-D-galactopyranosid e, or melibiose. Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to Delta pH. Overall, the se results are consistent with the notion that an acidic residue at po sition 325 is required for H+ transport via the lactose permease. Alte rnative hypotheses are also discussed.