PMID:1527014

Citation	Sirisena, DM, Brozek, KA, MacLachlan, PR, Sanderson, KE and Raetz, CR (1992) The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide. J. Biol. Chem. 267:18874-84
Abstract	We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.
Links	PubMed
Keywords	Amino Acid Sequence; Base Sequence; Biological Transport; Blotting, Southern; Chromosome Mapping; Cloning, Molecular; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Gene Expression; Genes, Bacterial; Heptoses/metabolism; Lipopolysaccharides/metabolism; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Salmonella typhimurium/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SALTY:RFAC		GO:0009244: lipopolysaccharide core region biosynthetic process	ECO:0000315:		P		Figure 5 - LPS profile of strains by SDS-PAGE. Mutant phenotype of ∆rfaC (lane 4) was restored to wildtype by complementing with an rfaC plasmid (lane 5).	complete CACAO 4999
SALTY:RFAC	involved_in	GO:0009244: lipopolysaccharide core region biosynthetic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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