Genetic and molecular studies of fitC4 and its suppressors fitA76* and fit95 in Escherichia coli.

Abstract

The fitA/pheS and fitB/pheT genes were previously proposed to function as transcription factors. The originally identified temperature sensitive (Ts) transcription-defective fitA76 mutant was shown to harbour a second mutation, fit95 (pheT) in addition to pheS5 (pheS; G₂₉₃ → A₂₉₃ transition). A new fit mutation namely, fitC4 (fitC locus) was identified in a Ts⁺ derivative of fitA76, namely JV4. Genetic mapping revealed that fitC4 mutation could be an extragenic suppressor, as it mapped at 39.01 min while fitAB loci mapped at 38.7 min on E. coli chromosome. Upon separation from JV4, fitC4 (Ts) failed to suppress the original fitA76 mutant (pheS5-fit95). Instead, JV4 harboured a modified form of fitA76 designated fitA76* (pheS4-fit95) with G₂₉₃ → C₂₉₃ transversion occurred at the same site of pheS5. The fitC4 and fitA76* mutations were genetically separated and reassembled to show that they both suppress each other as like in JV4. The separated fitC4 and fitA76* mutations behave like original fitA76 mutant in terms of transcription abnormality. This study focusses on further characterization of fitC4 and its accompanied mutations. The mutations fitC4, fitA76* and fitC4-fitA76* (reconstructed) are mobilized into new genetic backgrounds where the viability of these strains varied significantly. Growth and transcription abnormalities of fitC4 and fitA76* at 42 °C are restored in the reconstructed strain (fitC4-fitA76*), but not the β-galactosidase induction. As direct evidence, fit95 is shown to suppress fitC4 in a rpoB201 mutation background where fit95 phenotype is completely stabilized. The implications of these results with reference to transcription control by Fit factors in vivo are discussed.