Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C).
The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.
Sapunaric
Frederic M
FM
Center for Adaptation Genetics and Drug Resistance and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Levy
Stuart B
SB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics*; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D012694:Serine / Q000235:genetics*; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000378:metabolism; D013753:Tetracycline Resistance / Q000235:genetics* / Q000502:physiology
D001426:Bacterial Proteins; D012097:Repressor Proteins; C107173:TetC protein, Bacteria; D012694:Serine; D010649:Phenylalanine; D013752:Tetracycline