The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide.
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.
Sirisena
D M
DM
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Brozek
K A
KA
MacLachlan
P R
PR
Sanderson
K E
KE
Raetz
C R
CR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005798:Genes, Bacterial; D006539:Heptoses / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012486:Salmonella typhimurium / Q000235:genetics*
D004269:DNA, Bacterial; D006539:Heptoses; D008070:Lipopolysaccharides