Identification of a gene for aerobic growth with a SoxS binding sequence in Escherichia coli by operon fusion techniques

Eight Escherichia coli cells with aerobic growth defects were isolated by the insertion of λplacMu53, a hybrid bacteriophage of λ and Mu, which created transcriptional fusion to lacZY. Two of these mutants, CL10 and CL12, were irradiated with UV to obtain specialized transducing phages. The phages that took out the neighboring chromosomal DNA of the related gene responsible for defective aerobic growth were identified. The in vivo cloned chromosomal sequence revealed that the mutated gene of CL10 was located at min 34.5 on the Escherichia coli linkage map and 1,599,515 on the physical map. The physical map indicated that there were 7 cistrons in the operon. We named this operon oxg10. The promoter sequence of oxg10 exhibited a possible binding site for SoxS, a transcriptional regulator that activates the transcription of various SoxRS regulon genes. Transferring the oxg10::λplacMu53 mutation into the wild-type strain, RZ4500, resulted in the inhibition of normal aerobic growth, while the same mutation in strain MO inhibited aerobic cell growth completely. The full operon sequences of oxg10 were cloned from the Escherichia coli genomic library. The mutated gene of CL12 was identified to be a sucA gene encoding the α-ketoglutarate dehydrogenase E1 component in the TCA cycle.