Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome

Abstract

The nucleotide sequence of two kilobase pairs (kb) 5' to the orfE-pyrE operon has been determined. The sequence revealed two open reading frames, orfX and orfY, consisting of 286 and 274 codons, respectively, and having a transcriptional orientation opposite that of the orfE-pyrE operon. Analysis of transcription initiations showed that the promoters of orfE and orfX constitute a pair of divergent promoters with overlapping -35 regions and that orfY is transcribed from an independent promoter. Translational analysis indicated that the orfs are expressed in Escherichia coli. The orfE, orfX, and orfY genes were inactivated on the bacterial chromosome by deletion-insertion mutagenesis using a kanamycin resistance cassette. The mutants were all viable. However, the orfE deletion caused a dramatic reduction in the level of pyrE expression and a partial pyrimidine requirement, because this mutation prevented transcription of pyrE. the orfE protein seemed without significance for pyr-gene expression in E. coli, and the mutations in orfX and orfY were without detectable phenotypes.