The intermediate steps in the biosynthesis of the @entity857 - @entity11871 precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of @entity2624 . The mutation was in a gene homologous to the previously reported rfaE gene from @entity327 . The @entity526 rfaE 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 @entity535 sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I ( @entity1522 1 to 318) shared structural features with members of the ribokinase family, while Domain II ( @entity1522 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in @entity526 , as well as the rfaE543 mutation of @entity11621 SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one @entity535 substitution and a 12-bp in-frame deletion resulting in the loss of four @entity1522 , while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the @entity526 Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of @entity68608 , whereas Domain II catalyzes the @entity857 transfer to form @entity857 - @entity11871 .

The rfaE gene from XXXX encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose.

@entity1522 :: ('CHEBI:33709', 'Chemical') :: ['amino acids'] @entity11871 :: ('MESH:C026312', 'Chemical') :: ['D-glycero-D-manno-heptose', 'L-glycero-D-manno-heptose'] @entity535 :: ('CHEBI:33704', 'Chemical') :: ['amino acid'] @entity68608 :: ('CHEBI:60002', 'Chemical') :: ['D-glycero-D-manno-heptose 1-phosphate'] @entity327 :: ('727', 'Species') :: ['Haemophilus influenzae'] @entity11621 :: ('28901', 'Species') :: ['Salmonella enterica'] @entity857 :: ('MESH:D000244', 'Chemical') :: ['ADP'] @entity2624 :: ('83333', 'Species') :: ['Escherichia coli K-12'] @entity526 :: ('562', 'Species') :: ['E. coli']
@entity526 :: (562,Species) :: ['Escherichia coli']
