Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster.

Abstract

The genetic determinants of enterobacterial common antigen (ECA) include the rfe and r f f genes located between ilv and cya near min 86 on the Escherichia coli chromosome.The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79.The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, r f f T , and r f f M genes.Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffDlrffE-rffAl r f f C -r f f f -r f f M .The rfe gene was localized to a 2.54-kilobase CZuI fragment of DNA, and the complete nucleotide sequence of this fragment was determined.The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA.The putative initiation codon of ORF-1 was found to be 670 nucleotides downstream from the termination codon of rho.ORF-1 and ORF-2 specify putative proteins of 267 and 348 amino acids with calculated M, values of 29,010 and 39,771, respectively.ORF-1 was identified as the r f e gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli.Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-G1cNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.