DECREASED FUNCTION OF THE CLASS-B TETRACYCLINE EFFLUX PROTEIN TET WITH MUTATIONS AT ASPARTATE-15, A PUTATIVE INTRAMEMBRANE RESIDUE

The aspartate 15 residue within the first predicted intramembrane helix of the tetracycline efflux protein Tet has been conserved in four tetracycline resistance determinants from gram-negative bacteria. Its replacement in class B Tet by tyrosine, histidine, or asparagine resulted in a 60 to 85% loss of tetracycline resistance and a similar loss of tetracycline-proton antiport. The tyrosine and histidine substitutions lowered the V(max) of the efflux system by some 90% but did not alter the K(m). The asparagine substitution raised the K(m) over 13-fold, while the V(max) was equal to or greater than that of the wild type. Therefore, although the nature of its role is unclear, aspartate 15 is important for normal Tet function.