<jats:title>ABSTRACT</jats:title> <jats:p> The intermediate steps in the biosynthesis of the ADP- <jats:sc>l</jats:sc> - <jats:italic>glycero</jats:italic> - <jats:sc>d</jats:sc> - <jats:italic>manno</jats:italic> -heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn <jats:italic>10</jats:italic> insertion that confers a heptoseless LPS phenotype in the chromosome of <jats:italic>Escherichia coli</jats:italic> K-12. The mutation was in a gene homologous to the previously reported <jats:italic>rfaE</jats:italic> gene from <jats:italic>Haemophilus influenzae</jats:italic> . The <jats:italic>E. coli rfaE</jats:italic> gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the <jats:italic>aut</jats:italic> gene product of <jats:italic>Ralstonia eutrophus</jats:italic> . Each domain was expressed individually, demonstrating that only Domain I could complement the <jats:italic>rfaE</jats:italic> ::Tn <jats:italic>10</jats:italic> mutation in <jats:italic>E. coli</jats:italic> , as well as the <jats:italic>rfaE543</jats:italic> mutation of <jats:italic>Salmonella enterica</jats:italic> SL1102. DNA sequencing of the <jats:italic>rfaE543</jats:italic> gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the <jats:italic>aut</jats:italic> ::Tn <jats:italic>5</jats:italic> mutation in <jats:italic>R. eutrophus</jats:italic> is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the <jats:italic>E. coli</jats:italic> Domain II. Thus, both domains of <jats:italic>rfaE</jats:italic> are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of <jats:sc>d</jats:sc> - <jats:italic>glycero</jats:italic> - <jats:sc>d</jats:sc> - <jats:italic>manno</jats:italic> -heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP- <jats:sc>d</jats:sc> - <jats:italic>glycero</jats:italic> - <jats:sc>d</jats:sc> - <jats:italic>manno</jats:italic> -heptose.