ANTIBIOTIC-RESISTANCE MECHANISMS OF MUTANT EF-TU SPECIES IN ESCHERICHIA-COLI

Analysis of antibiotic-resistant EF-Tu mutants has revealed a connecti on between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EFTu resistant to kirromy cin (kir(r)) or pulvomycin (pul(r)). All kir(r) mutations cluster in t he interface of domains 1 and 3 of EF-Tu in its GTP-bound conformation , not in that of EF-Tu . GDP Other evidence also suggests that kirromy cin binds to the interface of wild-type EF-Tu, thereby jamming the GTP ase switch. Various functional studies reveal two subsequent resistanc e mechanisms. The first hinders kirromycin binding to EF-Tu . GTP and the second occurs after GTP hydrolysis by rejection of bound kirromyci n. All pul(r) mutations cluster in the three-domain junction interface of EF-Tu . GTP (which is an open hole in EF-Tu . GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tR NA binding. Mutations show that a D99-R230 salt bridge is not essentia l for the transduction of the GTPase switch signal from domain 1. In v ivo and in vitro studies reveal that pulvomycin sensitivity is dominan t over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may suppor t a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with in teresting effects on translational accuracy. Kir(r) EF-Tu(A375T) is ab le to reverse the streptomycin-dependent phenotype of a ribosomal prot ein S12 mutant strain to streptomycin sensitivity.