Resistance to the Antimicrobial Peptide Polymyxin Requires Myristoylation of Escherichia coli and Salmonella typhimurium Lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrAC strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3′-acyloxyacyl-linked myristate (C14) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group. Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrAC strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3′-acyloxyacyl-linked myristate (C14) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-d-manno-octulosonic acid; l-Ara4N, 4-amino-4-dexoy-l-arabinose; pEtN, phosphoethanolamine; ArnT, l-4-aminoarabinose transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. is the major surface molecule of Gram-negative bacteria and is held in the outer membrane by a unique phospholipid domain known as lipid A. The typical lipid A backbone consists of a β-1′,6-linked disaccharide of glucosamine that is phosphorylated and multiply acylated. In Escherichia coli and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), the disaccharide backbone is acylated at the 2-, 3-, 2′-, and 3′-positions with (R)-3-hydroxymyristate and phosphorylated at the 1- and 4′-positions (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). A secondary lauroyl (C12) and myristoyl (C14) group is attached at the 2′- and 3′-positions, respectively, of the distal glucosamine in an acyloxyacyl linkage resulting in the hexa-acylated structure shown in Fig. 1A (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). The lipid A domain is attached to the polysaccharide portion of LPS via the Kdo (3-deoxy-d-manno-octulosonic acid) sugars (Fig. 1) (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). Modification of the lipid A domain of E. coli and S. typhimurium with the cationic sugar 4-amino-4-dexoy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) promotes resistance to the cyclic antimicrobial lipopeptide, polymyxin (2.Helander I.M. Kilpelainen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, 3.Nummila K. Kilpelainen I. Zahringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (174) Google Scholar, 4.Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (245) Google Scholar, 5.Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar). Although the mechanism of polymyxin killing is not completely understood, the peptide is thought to access the outer surface of the bacterium by interacting with the negatively charged phosphate groups of lipid A. A similar mechanism is employed by cationic antimicrobial peptides of the innate immune system (6.Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). Masking of lipid A phosphate groups with positively charged amine-containing residues is predicted to decrease binding of polymyxin to the bacterial surface promoting survival. In E. coli and S. typhimurium, the polymyxin-resistant phenotype is primarily under the control of the PmrA/PmrB two-component regulatory system that is activated during growth under conditions of low pH, high Fe3+, and in a PhoP/PhoQ-dependent manner during Mg2+ starvation (7.Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 8.Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (340) Google Scholar, 9.Groisman E.A. J. Bacteriol. 2001; 183: 1835-1842Crossref PubMed Scopus (658) Google Scholar). Previously, Trent and co-workers (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) demonstrated that periplasmic addition of l-Ara4N to lipid A is catalyzed by l-4-aminoarabinose transferase (ArnT), a PmrA-regulated glycosyltransferase (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Because the transferase utilizes an undecaprenyl-linked donor substrate, undecaprenyl-phosphate-α-l-Ara4N, its active site is predicted to lie in the periplasmic region of the cell (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Furthermore, Doerrler et al. (12.Doerrler W.T. Gibbons H.S. Raetz C.R. J. Biol. Chem. 2004; : 45102-45109Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) have demonstrated that modification of lipid A with l-Ara4N and pEtN is dependent upon its transport across the inner membrane by MsbA. We now report that, in E. coli K12 and S. typhimurium, addition of l-Ara4N to the lipid A domain of LPS in living cells is dependent upon the presence of the acyloxyacyl-linked myristoyl group at the 3′-position. Loss of myristoylation of lipid A in both E. coli and S. typhimurium by inactivation of lpxM resulted in loss of l-Ara4N modification and in a significant decrease in polymyxin resistance. However, the pEtN modification of the lipid A phosphate groups was not effected by loss of myristoylation. Chemicals and Other Materials—[γ-32P]ATP and 32Pi were obtained from Amersham International. Silica Gel 60 (0.25-mm) thin layer plates were purchased from EM Separation Technology (Merck). Yeast extract and Tryptone were from Difco. Triton X-100 and bicinchoninic acid were from Pierce. Polymyxin B sulfate was purchased from Sigma. All other chemicals were reagent grade and were purchased from either Sigma or Mallinckrodt. Bacterial Strains and Growth Conditions—Bacterial strains are described in Table I. Typically bacteria were grown at 37 °C in LB broth containing 10 g of NaCl, 10 g of Tryptone, and 5 g of yeast extract per liter. When required for plasmid selection, cells were grown in the presence of 100 μg/ml ampicillin, 12 μg/ml μg/ml or μg/ml and or or coli M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. J. Bacteriol. 1992; PubMed Scopus Google S. Zahringer U. H. Mol. Microbiol. 1998; PubMed Scopus Google S. Zahringer U. H. Mol. Microbiol. 1998; PubMed Scopus Google of containing S. typhimurium PubMed Scopus Google containing E. coli in a were the were obtained from were purchased from was the were from the Gel and were purchased from All were used to the of mutants were generated by of the E. coli strain, WD101. a polymyxin-resistant E. coli K12 strain, a in the resulting in a pmrAC phenotype promoting polymyxin resistance (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). E. coli was with μg/ml of the for 10 at 37 that were to cells were grown LB plates mutants were by LB plates containing μg/ml polymyxin B sulfate at 37 were and sensitivity to polymyxin were by the and polymyxin-sensitive mutants were strains and of E. coli was generated by of the polymyxin-resistant of the E. coli lpxM Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. J. Bacteriol. 1992; PubMed Scopus Google Scholar). of and E. coli lpxM was the site of the PubMed Scopus Google Scholar) resulting in plasmid The was used to the pmrAC E. coli lpxM The S. typhimurium lpxM plasmid was by the Salmonella lpxM a The resulting plasmid was and was used to the S. typhimurium lpxM and of A lipid A was from cells with of 32Pi in 5 of LB broth as described Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The lipid A domain was from LPS by of the Kdo and by the of and co-workers Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were a Silica Gel 60 and in the were by and of were with 32Pi as described The containing the was and by as described by Trent and co-workers (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). of and of E. coli or S. typhimurium were grown at 37 °C to an of and by at g for All were at and were as described M.S. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) and were in at was by the bicinchoninic acid Biochem. PubMed Scopus Google as the of was generated from 100 of and the lipid the in of E. coli M.S. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google as described M.S. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of E. coli was as described by Trent and co-workers as the (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). of the l-Ara4N was as a to a lipid C.R. S. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Polymyxin coli and S. typhimurium strains were for polymyxin resistance as described by and Miller J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (340) Google Scholar). growth to an of bacteria were to in LB were for at 37 °C with of polymyxin in a to 100 of bacteria were LB plates and at 37 were for of polymyxin and the were as the of resulting from of coli or S. typhimurium were in of LB at 37 °C or containing 10 The were to an by at g for and with The cell were in of A was from cells and as described Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Cotter R.J. L. S. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and at to lipid A were by as described Cotter R.J. L. S. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I.M. L. K. Raetz C.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of A of the were in the a matrix-assisted laser of with a laser The was and a of for with the of 100 laser in and as the The were in and the by an portion of The was at °C to and of of E. coli of the amine-containing l-Ara4N and pEtN, to the phosphate groups of lipid A (Fig. 1) is with increased resistance to cationic antimicrobial the peptide polymyxin (2.Helander I.M. Kilpelainen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, 3.Nummila K. Kilpelainen I. Zahringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (174) Google Scholar, 4.Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (245) Google Scholar, 5.Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar). E. coli a in the resulting in a pmrAC phenotype and modification of the lipid A structure with l-Ara4N and pEtN, to resistance to polymyxin at of to μg/ml (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). we to additional modifications to the lipid A structure that were important for polymyxin resistance. as the strain, a of mutants sensitivity to polymyxin at μg/ml was generated via were LB containing μg/ml of and sensitivity to the peptide were for was in the presence of and the lipid A by Fig. the modification in the lipid A from polymyxin-sensitive mutants. lipid A were the polymyxin-sensitive mutants. mutants produced lipid A to that of E. coli a hexa-acylated lipid A phosphate groups (Fig. 1) and sensitivity to polymyxin at μg/ml not produced lipid A (Fig. to of the (Fig. with l-Ara4N, and pEtN sensitivity to the peptide at that other in polymyxin resistance to E. coli as in sensitivity from the to synthesize or (Fig. produced lipid A with or pEtN produced with l-Ara4N The in the lipid A domain of and from of l-Ara4N or pEtN transferase not strains and produced lipid A that not and for strains were for Strains and the to and l-Ara4N to of l-Ara4N to lipid A in the periplasmic region of the cell catalyzed by the inner membrane The utilizes an undecaprenyl-phosphate-α-l-Ara4N, as the donor (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Loss of or the to synthesize the undecaprenyl-linked in loss of polymyxin resistance J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar, J.S. Miller S.I. 2000; PubMed Scopus Google Scholar). Because mutants and sensitivity to we mutants the necessary enzymatic machinery to synthesize and l-Ara4N to the lipid A domain of The phospholipid from cells was by for the presence of the donor shown by Trent et al. (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google the l-Ara4N donor lipid was produced by the polymyxin-resistant (Fig. in the polymyxin-sensitive strain, However, of the phospholipid from mutants and (Fig. and the presence of the l-Ara4N donor Furthermore, mutants and from either or catalyzed the of l-Ara4N from the donor to (Fig. and a of lipid A. we were to that a decrease in polymyxin resistance in strains and not from the to or l-Ara4N to lipid A. of A by and by lipid A of mutants or were as described and upon Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Cotter R.J. L. S. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the lipid A containing phosphate groups the presence of a as by at in the The major at (Fig. to the loss of myristate (C14) from E. coli lipid A that is in E. coli mutants a S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. 1996; PubMed Scopus Google Scholar). The of the lipid was with either or pEtN groups as by major at (Fig. and (Fig. mutants or were to lipid A with l-Ara4N even though both mutants the necessary enzymatic machinery required for of l-Ara4N to lipid A Fig. The additional modification was addition of to containing either or pEtN groups at and (Fig. of the group is catalyzed by the outer membrane Gibbons H.S. Trent M.S. Miller S.I. Raetz C.R. J. 2000; PubMed Scopus (282) Google Scholar, L. Lim K.B. M. J.S. Hackett M. Miller S.I. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). were that the increased polymyxin sensitivity by mutants or resulted from a loss of l-Ara4N that myristoylation of E. coli lipid A in is a for l-Ara4N and of a E. coli loss of l-Ara4N modification in mutants and from a loss of addition of myristate to lipid we the E. coli A of polymyxin-resistant was used to the pmrAC an E. coli lpxM Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. J. Bacteriol. 1992; PubMed Scopus Google Scholar). The resulting was to as of the mutants and strains were with and the lipid A to modifications in the polymyxin-sensitive strains (Fig. and (Fig. 1) produced or hexa-acylated lipid The lipid A shown in 5 from were to with of from mutants (Fig. or not of with a E. coli resulted in of lipid A to that in the polymyxin-resistant was not the was with the of the lipid A of E. coli by produced to shown in Fig. for the polymyxin-sensitive not produced lipid A both myristate and However, the lipid A of produced major at and to hexa-acylated lipid A with l-Ara4N (Fig. Table a of the lipid A of strains and by of lipid A by A coli were obtained for E. coli strains and coli typhimurium typhimurium were obtained for E. coli strains and in a of S. typhimurium A of myristoylation of LPS to the of l-Ara4N addition to the lipid A domain of E. coli we or not the was for S. typhimurium an lpxM of S. typhimurium S. Zahringer U. H. Mol. Microbiol. 1998; PubMed Scopus Google we the of addition in bacteria containing a control or an lpxM plasmid Salmonella were in under conditions to the two-component regulatory Salmonella shown to of J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar, 8.Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (340) Google Scholar, L.F. M.M. Groisman E.A. J. 2000; PubMed Scopus Google to increased of lipid A phosphate groups with l-Ara4N and pEtN J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar, J.S. Miller S.I. 2000; PubMed Scopus Google Scholar). Previously, and co-workers S. Zahringer U. H. Mol. Microbiol. 1998; PubMed Scopus Google Scholar) demonstrated that the S. typhimurium lpxM produced an LPS an decrease in the of myristate However, the to phosphate groups were with l-Ara4N was not of lipid A from lpxM not or lpxM containing the by a major at to lipid A (Fig. The Salmonella lpxM a hexa-acylated lipid A with a major at (Fig. A and However, the at to a lipid A containing myristate The lpxM produced lipid A with at and (Fig. Table of l-Ara4N to the lipid A of E. coli was not by However, a at (Fig. to the addition of a l-Ara4N sugar to lipid A of the Salmonella was present. of the Salmonella with the lpxM plasmid resulted in the of lipid A in Salmonella grown under conditions L. Lim K.B. M. J.S. Hackett M. Miller S.I. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Lim K.B. J.S. Hackett M. Miller S.I. 276: PubMed Scopus Google Scholar). The of lipid A containing l-Ara4N-modified was increased as with the Salmonella lpxM with major at and (Fig. and a with l-Ara4N sugars was as by the at Table The of lipid A from the was by the of the lipid A by the addition of from the of a lipid A required for the addition of myristate to S. typhimurium lipid A Table of the Polymyxin of E. coli and S. typhimurium coli a in to a pmrAC phenotype resulting in increased resistance to polymyxin. by the polymyxin-resistant phenotype is upon loss of a of lpxM Table A similar was the of E. coli with of polymyxin in shown in Fig. E. coli a in polymyxin sensitivity with its pmrAC strain, WD101. of with the resulted in of polymyxin resistance to the by and Fig. Salmonella lpxM mutants grown under conditions increased sensitivity to polymyxin that by of an lpxM plasmid not polymyxin sensitivity of E. coli an lpxM are described in Table of of of to resistance and is to the of the Strains are described in Table A of of to resistance and is to the of the in a In E. coli and S. typhimurium, the of the lipid A domain of LPS is by the addition of myristate catalyzed by the (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). as the for the Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the to the of the inner A attached to the is across the inner membrane by the 2001; PubMed Scopus Google Scholar, W.T. Raetz C.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). transport to the periplasmic lipid A with the cationic sugar l-Ara4N (12.Doerrler W.T. Gibbons H.S. Raetz C.R. J. Biol. Chem. 2004; : 45102-45109Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The is from (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) by the inner membrane to the phosphate groups of lipid A (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). of l-Ara4N to the lipid A domain of Gram-negative bacterial LPS to resistance to cationic antimicrobial such as polymyxin (2.Helander I.M. Kilpelainen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, J.S. Miller S.I. 2000; PubMed Scopus Google Scholar, M. Vaara M. Helander I. M. PubMed Scopus Google Scholar, J.S. PubMed Scopus Google Scholar). In an attempt to lipid A modifications necessary for polymyxin resistance, we polymyxin-sensitive mutants of an E. coli K12 pmrAC A subset of mutants to lipid A domain with l-Ara4N to myristate at the 3′-position. were by an E. coli a of the pmrAC to the that in modification of LPS with l-Ara4N myristoylation of the lipid A However, we necessary to the were for S. E. coli and S. typhimurium synthesize a hexa-acylated lipid A with the transport across the We the lipid A from S. typhimurium, containing an lpxM grown under conditions known to modification to the lipid A When the Salmonella lpxM was under lipid A with pEtN were by a of the lipid A was with l-Ara4N from the S. typhimurium lpxM (Fig. A and However, upon of the Salmonella lpxM the an in l-Ara4N-modified lipid A were by the bacteria produced lipid A containing l-Ara4N attached to both phosphate groups of its lipid A (Fig. and l-Ara4N is attached via a linkage to the of E. coli and S. typhimurium lipid A Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Ribeiro A.A. Lin S. Cotter R.J. Miller S.I. Raetz C.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Ribeiro A.A. Raetz C.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in S. typhimurium a portion of the lipid A l-Ara4N (Fig. 1) Ribeiro A.A. Lin S. Cotter R.J. Miller S.I. Raetz C.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We that inactivation of lpxM in E. coli in a loss of l-Ara4N addition, the Salmonella lpxM produced a of l-Ara4N-modified lipid A. Previously, Trent and co-workers (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) demonstrated that in the l-Ara4N transferase of Salmonella l-Ara4N to the group the lipid A lipid as the However, is used as the in the system sugars to the molecule (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Because S. typhimurium mutants with l-Ara4N at the C.R. S. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, I.M. L. K. Raetz C.R. J. Biol. Chem. Full Text PDF PubMed Google was that modification of the group is dependent upon the presence of the Kdo (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). is that the of l-Ara4N in the Salmonella lpxM is linked to the group. upon of l-Ara4N to the group of lipid A in both the presence of the Kdo and Because is in the periplasmic region of the is that the a hexa-acylated lipid A domain for A of the of required to the of Other lipid A have shown to to for et al. C. A. S. Cotter Trent S. J. Bacteriol. PubMed Scopus Google Scholar) demonstrated that an Kdo at the by an H. lipid A for the outer membrane of to activated in the of l-Ara4N modification K. Miller S.I. J. Bacteriol. PubMed Scopus Google Scholar). modification of LPS the Gram-negative membrane at the enzymatic the we have an additional phenotype of lpxM mutants. 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