title: Escherichia coli mutants that synthesize dephosphorylated lipid A molecules.
abstract: The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine that is phosphorylated at the 1 and 4' positions. Expression of the Francisella novicida lipid A 1-phosphatase FnLpxE in E. coli results in dephosphorylation of the lipid A proximal unit. Coexpression of FnLpxE and the Rhizobium leguminosarum lipid A oxidase RlLpxQ in E. coli converts much of the proximal glucosamine to 2-amino-2-deoxygluconate. Expression of the F. novicida lipid A 4'-phosphatase FnLpxF in wild-type E. coli has no effect because FnLpxF cannot dephosphorylate hexaacylated lipid A. However, expression of FnLpxF in E. coli lpxM mutants, which synthesize pentaacylated lipid A lacking the secondary 3'-myristate chain, causes extensive 4'-dephosphorylation. Coexpression of FnLpxE and FnLpxF in lpxM mutants results in massive accumulation of lipid A species lacking both phosphate groups, and introduction of RlLpxQ generates phosphate-free lipid A variants containing 2-amino-2-deoxygluconate. The proposed lipid A structures were confirmed by electrospray ionization mass spectrometry. Strains with 4'-dephosphorylated lipid A display increased polymyxin resistance. Heptose-deficient mutants of E. coli lacking both the 1- and 4'-phosphate moieties are viable on plates but sensitive to CaCl(2). Our methods for reengineering lipid A structure may be useful for generating novel vaccines and adjuvants.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Ingram
authors.forename: Ali
authors.initials: A
authors.lastname: Masoudi
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005603:Francisella / Q000235:genetics / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D008050:Lipid A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011113:Polymyxins / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D029968:Escherichia coli Proteins; D007477:Ions; D008050:Lipid A; D008070:Lipopolysaccharides; D011113:Polymyxins; C095015:lipid A 4'-phosphatase; D010744:Phosphoric Monoester Hydrolases
references.citation: J Biol Chem. 2004 Nov 19;279(47):49470-8
references.pmid: 15339914
references.citation: J Am Chem Soc. 2003 May 21;125(20):6103-12
references.pmid: 12785841
references.citation: J Biol Chem. 1997 Apr 18;272(16):10353-60
references.pmid: 9099672
references.citation: Mol Microbiol. 1998 Mar;27(6):1171-82
references.pmid: 9570402
references.citation: J Biol Chem. 1994 May 20;269(20):14402-10
references.pmid: 8182046
references.citation: J Biol Chem. 1987 Sep 15;262(26):12570-4
references.pmid: 2442165
references.citation: Science. 2007 Jun 15;316(5831):1628-32
references.pmid: 17569868
references.citation: Anal Biochem. 1985 Oct;150(1):76-85
references.pmid: 3843705
references.citation: J Bacteriol. 1992 Feb;174(3):702-10
references.pmid: 1732206
references.citation: J Biol Chem. 2006 Apr 7;281(14):9321-30
references.pmid: 16467300
references.citation: Annu Rev Biochem. 2007;76:295-329
references.pmid: 17362200
references.citation: Biochemistry. 2008 Aug 19;47(33):8623-37
references.pmid: 18656959
references.citation: J Biol Chem. 2001 Nov 16;276(46):43111-21
references.pmid: 11535603
references.citation: J Biol Chem. 2000 Sep 8;275(36):28017-27
references.pmid: 10856304
references.citation: J Biol Chem. 2006 May 5;281(18):12879-87
references.pmid: 16497671
references.citation: J Biol Chem. 2001 Nov 16;276(46):43132-44
references.pmid: 11535605
references.citation: J Biol Chem. 2002 Apr 19;277(16):14194-205
references.pmid: 11830595
references.citation: Chemistry. 2004 Oct 4;10(19):4798-807
references.pmid: 15372693
references.citation: J Biol Chem. 2009 Jun 5;284(23):15369-89
references.pmid: 19346244
references.citation: Can J Biochem Physiol. 1959 Aug;37(8):911-7
references.pmid: 13671378
references.citation: Nat Rev Microbiol. 2009 Sep;7(9):677-83
references.pmid: 19633680
references.citation: J Biol Chem. 2003 Oct 10;278(41):39269-79
references.pmid: 12869541
references.citation: Expert Rev Vaccines. 2007 Apr;6(2):133-40
references.pmid: 17408363
references.citation: J Biol Chem. 2004 Oct 22;279(43):45102-9
references.pmid: 15304478
references.citation: Biochim Biophys Acta. 2009 Jul;1791(7):594-602
references.pmid: 19416651
references.citation: Biochemistry. 2006 Dec 5;45(48):14427-40
references.pmid: 17128982
references.citation: Expert Opin Biol Ther. 2004 Jul;4(7):1129-38
references.pmid: 15268679
references.citation: J Immunol. 1989 May 1;142(9):3229-38
references.pmid: 2651523
references.citation: J Clin Invest. 1996 Jan 15;97(2):359-65
references.pmid: 8567955
references.citation: Nature. 2009 Apr 30;458(7242):1191-5
references.pmid: 19252480
references.citation: J Biol Chem. 2002 Nov 1;277(44):41811-6
references.pmid: 12193596
references.citation: J Biol Chem. 2009 Jan 2;284(1):414-425
references.pmid: 18984595
references.citation: J Biol Chem. 2000 Sep 8;275(36):28006-16
references.pmid: 10856303
references.citation: Antimicrob Agents Chemother. 1999 Jun;43(6):1459-62
references.pmid: 10348770
references.citation: EMBO J. 2000 Oct 2;19(19):5071-80
references.pmid: 11013210
references.citation: J Biol Chem. 1982 Oct 10;257(19):11808-15
references.pmid: 6749846
references.citation: J Biol Chem. 2004 Dec 31;279(53):55780-91
references.pmid: 15489235
references.citation: J Biol Chem. 2003 Apr 4;278(14):12120-9
references.pmid: 12531908
references.citation: Microbiol Mol Biol Rev. 2003 Dec;67(4):593-656
references.pmid: 14665678
references.citation: J Biol Chem. 2006 May 5;281(18):12865-78
references.pmid: 16497674
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):4136-41
references.pmid: 17360489
references.citation: Eur J Biochem. 1997 Jul 15;247(2):716-24
references.pmid: 9266718
references.citation: Biochim Biophys Acta. 2010 May;1801(5):593-604
references.pmid: 20153447
references.citation: Gene. 1991 Apr;100:195-9
references.pmid: 2055470
references.citation: Science. 2007 Jun 15;316(5831):1574-6
references.pmid: 17569850
references.citation: J Biol Chem. 2001 Apr 13;276(15):11461-4
references.pmid: 11278265
references.citation: J Lipid Res. 2006 May;47(5):1097-111
references.pmid: 16479018
references.citation: FASEB J. 1994 Feb;8(2):217-25
references.pmid: 8119492
references.citation: J Biol Chem. 2005 Jun 3;280(22):21202-11
references.pmid: 15795227
references.citation: Chembiochem. 2006 Jan;7(1):140-8
references.pmid: 16317789
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19005-10
references.pmid: 18024585
references.citation: J Biol Chem. 1985 May 10;260(9):5271-8
references.pmid: 3988753
references.citation: Gene. 1990 Nov 30;96(1):23-8
references.pmid: 2265755
references.citation: J Biol Chem. 2005 Apr 8;280(14):14154-67
references.pmid: 15695810
references.citation: Int J Oral Sci. 2009 Sep;1(3):126-35
references.pmid: 20657724
references.citation: J Biol Chem. 2002 Nov 1;277(44):41802-10
references.pmid: 12193590
references.citation: J Biol Chem. 2002 Aug 9;277(32):28959-71
references.pmid: 12019272
references.citation: J Biol Chem. 2003 Apr 4;278(14):12109-19
references.pmid: 12531907
references.citation: Cell Microbiol. 2009 Nov;11(11):1587-99
references.pmid: 19552698