title: Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli.
abstract: Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection 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 EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT 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 tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.
authors.affiliation: Leiden Institute of Chemistry, Department of Biochemistry, Leiden University, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: Kraal
authors.forename: L A
authors.initials: LA
authors.lastname: Zeef
authors.forename: J R
authors.initials: JR
authors.lastname: Mesters
authors.forename: K
authors.initials: K
authors.lastname: Boon
authors.forename: E L
authors.initials: EL
authors.lastname: Vorstenbosch
authors.forename: L
authors.initials: L
authors.lastname: Bosch
authors.forename: P H
authors.initials: PH
authors.lastname: Anborgh
authors.forename: A
authors.initials: A
authors.lastname: Parmeggiani
authors.forename: R
authors.initials: R
authors.lastname: Hilgenfeld
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D010641:Phenotype; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D010444:Peptide Elongation Factor Tu