A growth-defective kirromycin-resistant EF-Tu Escherichia coli mutant and a spontaneously evolved suppression of the...

This study has investigated the cause of a growth-defect phenotype of a mutation in the elongation factor EF-Tu from Escherichia coli. An M13-based genetic retrieval system reported by Zeef and Bosch [Mol. Gen. Genet. 238 (1993) 252-260] was used to segregate and identify an extremely growth-defective kirromycin-resistant (KrR) tufA mutation, encoding Gln124-->Lys (Q124K), from a KrR parent strain. This original strain also contained mutations, 124com1 and 124com2, that appear to have evolved to suppress the Q124K tufA mutation. In this communication we present these M13-based genetic experiments together with additional genetic and protein characterization experiments to clarify the basis of this complementation. The data indicate that the serious growth defect of Q124K originates from a defective GTP/GDP interaction. The GTP/GDP binding and GTP hydrolysis characteristics of ET-Tu Q124K were different from wild-type EF-Tu and especially of another KrR EF-Tu mutant A375T. In line with this, 124com1 specifically complemented EF-Tu Q124K, whereas the growth defects of strains containing EF-Tu mutated at aa 375 were aggravated. We also show that strains containing the segregated tufA Q124K mutation formed filaments.