Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4'-phosphatase LpxF.
Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Xiaoyuan
X
Wang
Sara C
SC
McGrath
Robert J
RJ
Cotter
Christian R H
CR
Raetz
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane; D003001:Cloning, Molecular; D004926:Escherichia coli; D005603:Francisella / Q000235:genetics*; D015870:Gene Expression; D008050:Lipid A / Q000378:metabolism; D008969:Molecular Sequence Data; D019897:Periplasm; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000235:genetics*; D042163:Rhizobium etli / Q000235:genetics; D016961:Rhizobium leguminosarum / Q000235:genetics; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D008050:Lipid A; C095015:lipid A 4'-phosphatase; D010744:Phosphoric Monoester Hydrolases