Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325-->Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325-->Ser) plus His-322-->Asp. Seven revertants showed a second site mutation His-322-->Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325-->Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322, A site-directed double mutant His-322-->Asn/Glu-325-->Asn showed a greater rate of lactose uptake (V-max) than either of the single mutants His-322-->Asn or Glu-325-->Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.