Abstract
The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 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, rffT, and rffM 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-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI 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 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF-1 was identified as the rfe 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-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.
Highlights
Antigen (ECA) include the r f e and r f fgenes located between ilv and c y a near min 86 on the Escherichia coli chromosome
Recent studies from this laboratory have established that Enterobacterial common antigen (ECA)-negative rfe::TnlO-insertion mutants of E. coli are defective in lipid I synthesis (13).These observations suggestedthe possibility that the rfe gene codes for the transferase that catalyzes the transfer of GlcNAc 1
We report the cloning of the rfe gene on a 2.54kb ClaI fragmentof E. coli DNA
Summary
Recent studies from this laboratory have established that ECA-negative rfe::TnlO-insertion mutants of E. coli are defective in lipid I synthesis (13).These observations suggestedthe possibility that the rfe gene codes for the transferase that catalyzes the transfer of GlcNAc 1-. Restriction has allowed the construction of a partial map of the rfe-rff endonuclease digestions and DNA ligations were carried out as recregion. Construction of Cosmid Clone pCAll and Subclones-The cosmid cloning of E. coli DNA wascarried out using total chromosomal DNA from E. coli strain AN2618 that had been partially cut with the enzyme Sau3A. DNA fragments (20 pg) within the 34-49-kbsize rfe::TnlO-insertionmutants of E. coli, and the nucleotide range were ligated to the cosmid vector pHC79 (10 pg)following sequence of the putative rfe gene is presented. A portion of the ligated DNA (7.5 pg) was packaged encodes the UDP-G1cNAc:undecaprenylphosphate GlcNAc1-phosphate transferase responsiblfeor lipid I synthesis. The ECA-negative mutant strains 21546,21548, and 21550were each transduced with 1.5pgof the packaged DNA and ampicillin-resistant transductants were selected
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