AnUndecaprenylPhosphate-AminoarabinoseFlippase ... · AnUndecaprenylPhosphate-AminoarabinoseFlippase ... functions of the Pmr operon gene products in aminoarabinose (L-Ara4N) biosynthesis

An Undecaprenyl Phosphate-Aminoarabinose FlippaseRequired for Polymyxin Resistance in Escherichia coli*□S

Received for publication, July 26, 2007, and in revised form, September 17, 2007 Published, JBC Papers in Press, October 10, 2007, DOI 10.1074/jbc.M706172200

Aixin Yan1, Ziqiang Guan2, and Christian R. H. Raetz3

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Modification of lipid A with the 4-amino-4-deoxy-L-arabi-nose (L-Ara4N) moiety is required for resistance to polymyxinand cationic antimicrobial peptides in Escherichia coli and Sal-monella typhimurium. An operon of seven genes (designatedpmrHFIJKLM in S. typhimurium), which is regulated by thePmrA transcription factor and is also present in E. coli, is neces-sary for the maintenance of polymyxin resistance. We previ-ously elucidated the roles of pmrHFIJK in the biosynthesis andattachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively.Wenowpropose functions for the last twogenes of the operon, pmrL and pmrM. Chromosomal inactiva-tion of each of these genes in anE. coli pmrAcparent switched itsphenotype from polymyxin-resistant to polymyxin-sensitive.Lipid A was no longer modified with L-Ara4N, even though thelevels of the lipid-linked donor of the L-Ara4N moiety, undeca-prenyl phosphate-�-L-Ara4N, were not reduced in themutants.However, the undecaprenyl phosphate-�-L-Ara4N present inthemutants was less concentrated on the periplasmic surface ofthe inner membrane, as judged by 4–5-fold reduced labelingwith the inner membrane-impermeable amine reagent N-hy-droxysulfosuccin-imidobiotin. In an arnT mutant of the samepmrAc parent, which lacks the enzyme that transfers theL-Ara4N unit to lipid A but retains the same high levels of undeca-prenyl phosphate-�-L-Ara4N as the parent, N-hydroxysulfosuc-cinimidobiotin labeling was not reduced. These results implicatepmrLandpmrM,butnotarnT, in transportingundecaprenylphos-phate-�-L-Ara4N across the inner membrane. PmrM and PmrL,now renamed ArnE and ArnF because of their involvement inL-Ara4Nmodificationof lipidA,maybe subunits of anundecapre-nyl phosphate-�-L-Ara4N flippase.

The lipid A moiety of lipopolysaccharide (LPS)4 in the outermembranes of Gram-negative bacteria typically consists of a

�,1�–6-linked disaccharide of glucosamine, modified withphosphate groups at the 1- and 4�-positions, and with R-3-hy-droxyacyl chains at the 2-, 3-, 2�-, and 3�-positions (Fig. 1) (1, 2).Lipid A plays an important role in the pathogenesis of Gram-negative bacterial infections. It potently activates the innateimmune system (3, 4), triggering the synthesis of cationic anti-microbial peptides, cytokines, clotting factors, and otherimmunostimulatory molecules (5–8).Polymyxin B, a cationic antibiotic secreted by certain Gram-

positive bacteria, binds to lipid A with high affinity and dam-ages the cell envelope (9, 10). Mutants of Gram-negative bacte-ria resistant to polymyxin B usually acquire constitutivemutations in the transcription factor PmrA (11–13). The lipidA molecules isolated from these pmrAc strains are extensivelymodified with phosphoethanolamine (pEtN) and 4-amino-4-deoxy-L-arabinose (L-Ara4N) moieties (Fig. 1), which reducethe net negative charge of lipid A and limit its interaction withpolymyxin (14–21). Transcription of the genes encoding theenzymes responsible for lipid A modification with pEtN andL-Ara4N are under the direct control of PmrA (2, 22, 23), whichcan also be activated in wild-type Escherichia coli by externalstimuli, such as lowpH inphagolysosomes or exposure tometa-vanadate (24–26). Attachment of the L-Ara4N moiety (27) tolipid A is essential for resistance to polymyxin B and other cat-ionic antimicrobial peptides (11, 28).A Salmonella typhimurium operon of seven genes, designated

pmrHFIJKLM, is co-transcribed under the control of the tran-scription factor PmrA (16). The Pmr gene products are requiredfor themaintenance of polymyxin resistance and the attachmentof the L-Ara4Nmoiety to lipid A (16). The same operon is pres-ent in E. coli. Based upon the predicted sequences of the Pmrprotein products, we proposed a biosynthetic pathway for gen-erating the L-Ara4N moiety and transferring it to lipid A (25).We have previously assigned enzymatic functions to five(PmrHFIJK) of the seven gene products of the operon and haverenamed themArnB (29), ArnC (28), ArnA (28, 30), ArnD (28),and ArnT (13, 31), respectively, to reflect their roles in L-Ara4Nproduction (Fig. 2, A and B). The pathway starts with the con-version of UDP-glucose to UDP-glucuronic acid (Fig. 2A) (30).The C-terminal domain of ArnA then catalyzes the oxidativedecarboxylation of UDP-glucuronic acid to generate a novelUDP-4-ketopentose (28, 30, 32, 33). This substance is transam-inated byArnB (29, 34), using glutamic acid as the amine donor,to generate UDP-�-L-Ara4N, which is N-formylated by theN-terminal domain of ArnA (Fig. 2A) (28). ArnC next transfers

* This work was supported in part by National Institutes of Health GrantGM-51310 (to C. R. H. R.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1.

1 Present address: Dept. of Biomolecular Chemistry, University of Wisconsin,Madison, WI 53706.

2 Supported by the LIPID MAPS Large Scale Collaborative Grant GM-069338from the National Institutes of Health.

3 To whom correspondence should be addressed: Dept. of Biochemistry,Duke University Medical Center, P. O. Box 3711, Durham, NC 27710. Tel.:919-684-3384; Fax: 919-684-8885; E-mail: [email protected].

4 The abbreviations used are: L-Ara4N, 4-amino-4-deoxy-L-arabinose; ESI/MS,electrospray ionization/mass spectrometry; LPS, lipopolysaccharide; NHS-

biotin, N-hydroxysuccinimidobiotin; pEtN, phosphoethanolamine; Sulfo-NHS-biotin, N-hydroxysulfosuccinimidobiotin; ABC, ATP-binding cassette.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 49, pp. 36077–36089, December 7, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

DECEMBER 7, 2007 • VOLUME 282 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 36077

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the N-formylated L-Ara4N moiety to undecaprenyl phosphate,the product of which is rapidly deformylated by ArnD (Fig. 2A)(28). After being transported to the outer surface of the innermembrane by an unknown mechanism, the L-Ara4N group ofundecaprenyl phosphate-L-Ara4N is transferred to lipid A byArnT (13, 31) (Fig. 2A). Further evidence that L-Ara4N transferto lipid A occurs on the outer surface of the inner membrane isprovided by the observation that L-Ara4Nmodification of lipidA is dependent upon the essential core-lipid A flippase, MsbA(35, 36).We now report on the functions of the two remaining gene

products of the operon, PmrL and PmrM (Fig. 2B) (16), whichdisplay distant sequence similarity to the multidrug effluxpump exporter EmrE (37–39). In-frame chromosomal deletionmutants of pmrL and pmrM, generated in a polymyxin-resis-tant pmrAc parental strain, regain polymyxin sensitivity.Despite the presence of the same high levels of undecaprenylphosphate-�-L-Ara4N in these mutants as in their parent, lipidA modification with L-Ara4N is almost completely abolished.Using the inner membrane-impermeable amine reagent N-hy-droxysulfosuccin-imidobiotin (sulfo-NHS-biotin) (36), in con-junction with mass spectrometry, we show that undecaprenylphosphate-�-L-Ara4N is 4–5-fold less accessible to chemicalmodification in pmrL/pmrAc and pmrM/pmrAc mutants thanin the pmrAc parent, presumably reflecting the reduced ability

of undecaprenyl phosphate-�-L-Ara4N to gain access to theperiplasmic side of the inner mem-brane. Reduced chemical modifica-tion of undecaprenyl phosphate-�-L-Ara4N is not observed in an arnTmutant derived from the samepmrAc parent, in which undecapre-nyl phosphate-�-L-Ara4N levelsalso remain as high as they are in theparent. We suggest that PmrL andPmrM, renamed ArnE and ArnF,respectively (see Fig. 2B for the oldand new nomenclature), function asa heterodimer to flip undecaprenylphosphate-�-L-Ara4N from thecytosolic side to the periplasmicside of the inner membrane, wherethe active site of ArnT is located(Fig. 2A).

EXPERIMENTAL PROCEDURES

Materials—Glass-backed 0.25-mm Silica Gel 60 TLC plates werepurchased fromMerck. Chloroformwas obtained from EM Science,whereas pyridine, methanol, andformic acid were from Mallinck-rodt. Trypticase soy broth, yeastextract, and tryptone were pur-chased from Difco. Pfu PCRreagents were purchased fromStratagene, and Taq PCR reagents

were from Roche Applied Science. The spin miniprep and gelextraction kits were from Qiagen. Sulfo-NHS-biotin, a mem-brane-impermeable amine reagent (40), andN-hydroxysuccin-imidobiotin (NHS-biotin), a membrane-permeable analogue,were the products of Pierce.Bacterial Strains—The bacterial strains used in this study are

described in Table 1. Typically, bacteria were grown at 37 °C inLB medium, which consists of 10 g of NaCl, 10 g of tryptone,and 5 g of yeast extract per liter (41). When required for selec-tion of plasmids or mutations, cells were grown in the presenceof 100 �g/ml ampicillin, 10 �g/ml polymyxin, or 20 �g/mlkanamycin.Molecular Biology Applications—Protocols for handling of

DNA samples were those of Sambrook and Russell (42). Trans-formation-competent cells of E. coli were prepared by themethod of Inoue et al. (43). When required, E. coli cells wereprepared for electroporation by the method of Sambrook andRussell (42). Plasmids were prepared using the Qiagen spinprep kit. DNA fragments were isolated from agarose gels usingthe QIAquick gel extraction kit. Genomic DNA was isolatedusing the protocol for bacterial cultures in the Easy-DNATM kit(Invitrogen). The rapid DNA ligation kit (Roche Applied Sci-ence), restriction endonucleases (New England Biolabs), andshrimp alkaline phosphatase (U. S. Biochemical Corp.) wereused according to the manufacturer’s instructions. Double-

FIGURE 1. Regulated covalent modifications of lipid A in E. coli and S. typhimurium. As indicated by thedashed bonds, modifications of the phosphate groups and/or acyl chains of lipid A are observed under certaingrowth conditions or in some mutants (2). The phosphates can be modified with L-Ara4N and/or pEtN groups,both of which are regulated by pmrA (26). The latter gene, encoding the PmrA transcription factor (73), isconstitutively active in polymyxin-resistant mutants, and the relevant modifying enzymes ArnT (31) and EptA(22, 23) are induced under these conditions. In some instances, lipid A species are made in which the locationsof the L-Ara4N and pEtN groups are reversed (20, 88), or in which both phosphates are modified with the samegroup, as is the case with pEtN when L-Ara4N synthesis is blocked (21). The outer membrane enzyme PagP (89)can add an additional palmitate unit at position 2, whereas PagL (90) can remove the hydroxymyristoyl chainat position 3. These pag genes are regulated by the PhoP/PhoQ system (91), which can be activated by lowMg2� in Salmonella. PagL (90), LpxO (92), and LpxR (93) are present in Salmonella but not E. coli K-12. However,they can function in E. coli K-12 when expressed from plasmids.

Inner Membrane Transport of Undecaprenyl Phosphate-�-L-Ara4N

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stranded DNA sequencing was performed with an ABI Prism377 instrument at the Duke University DNA Analysis Facility.Primers were purchased fromMWG Biotec.In-frameReplacements of pmrL, pmrM, pmrLM, or arnTwith

Kanamycin Resistance Cassettes in a pmrAc Derivative of E. coliDY330—The old and new gene nomenclature is summarized inFig. 2B and Table 2. The pmr designations (11, 12, 16) wereoriginally used to describe genes required for the maintenanceof polymyxin resistance in S. typhimurium (Fig. 2B). A similargroup of genes, present in E. coli, was originally identified usingthe “Yfb,” “Orf,” or “b” nomenclatures (Table 2) (44, 45). TheArn system (28–30) should now be adopted, given that thefunctions of the Pmr operon gene products in aminoarabinose(L-Ara4N) biosynthesis are now well defined (Fig. 2A), includ-ing PmrL and PmrM, as described below.

The construction of an in-frame replacement of pmrLwith akanamycin resistance cassette (kan) was based on the methodof Derbise et al. (46). A pmrAc derivative of E. coli strain DY330(47), designated MST100 (28), which contains a � prophageharboring the recombination genes exo, bet, and gam undercontrol of a temperature-sensitive � cI-repressor, was utilizedas the parental strain.A linear piece of DNA consisting of a kan cassette flanked by

500 bp of E. coli DNA upstream and downstream of pmrL wasgenerated by a three-step overlap PCR procedure. First, E. coliK-12W3110 genomicDNAwas used as the template to amplifythe 500-bp upstream and downstream regions of pmrL. Theupstream segment included on its 3� end the first 20 bp of thekan gene. The downstream region included on its 5� end the last20 bp of the kan gene plus a ribosomal binding site. The forward

FIGURE 2. Biosynthesis of undecaprenyl phosphate-�-L-Ara4N and transfer of the L-Ara4N moiety to lipid A. A, in polymyxin-resistant E. coli and S.typhimurium, biosynthesis of the L-Ara4N moiety begins with oxidation of UDP-glucose to UDP-glucuronic acid (2). Next, the C-terminal domain of ArnAcatalyzes the NAD�-dependent oxidative decarboxylation of UDP-glucuronic acid to yield an unusual UDP-4-ketopentose (30, 32), which is convertedby the transaminase ArnB to UDP-�-L-Ara4N (29). Subsequently, the N-terminal domain of ArnA uses N-10-formyltetrahydrofolate to N-formylateUDP-�-L-Ara4N (28, 30, 32). ArnC (a distant orthologue of dolichyl phosphate-mannose synthase) selectively transfers the L-Ara4-formyl-N residue toundecaprenyl phosphate (28). Next, ArnD catalyzes deformylation of this substance to undecaprenyl phosphate-�-L-Ara4N (13), preventing the reversalof the ArnC-catalyzed reaction (28). After transport to the outer surface of the inner membrane, presumably by ArnE (PmrL) and ArnF (PmrM) as shownin the present study, the membrane enzyme ArnT (31) transfers the L-Ara4N moiety to lipid A. B, order of genes and direction of transcription (left to right)of the pmr operon (16). The preferred arn terminology (2, 45), which is consistent with the generalized bacterial polysaccharide gene nomenclature (94),is shown in red. We suggest that the older pmr nomenclature be retained for the regulatory genes pmrA, pmrB, and pmrD, because their products havemany other functions besides regulating the expression of enzymes needed for L-Ara4N biosynthesis.




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and reverse primers used for amplifying the upstream (PmrL-ufand PmrL-ur) and downstream 500-bp regions (PmrL-df andPmrL-dr) are provided in supplemental Table 1.In parallel, the kanamycin resistance gene (Tn903) fromplas-

mid pWKS130 (48) was utilized as the template to amplify thekan cassette, flanked on its 5� end by 20 bp of chromosomalDNA immediately upstream of pmrL, and flanked on its 3� endby a ribosome-binding site plus 20 bp of chromosomal DNAlocated immediately downstream of pmrL. The primers foramplifying this DNA fragment (Kan-PmrL-f and Kan-PmrL-r)are likewise provided in supplemental Table 1.In the second step, a mixture of 100 ng of each of the

upstream and downstream PCR products, together with 100 ngof the kan cassette PCR fragment, served as the templates toamplify a large piece of DNA containing the desired segment,consisting of the 500 bpupstreamofpmrL, the kan cassette, andthe 500 bp downstream of pmrL. The primers (PmrL-uf andPmrL-dr) used for this purpose are provided in supplementalTable 1.Becausemultiple bands were generated during this PCR pro-

cedure, a third step was introduced. The DNA fragment of thecorrect size was recovered from an agarose gel, and 50 ng of thegel-purified material was used as the template DNA for a sec-ond PCR with the same pair of the primers. The resulting PCRproduct was resolved on a 1.0% agarose gel and purified. The

product was then electroporated into MST100 cells. Colonieswere selected on LB agar containing kanamycin (20 �g/ml)after incubation overnight at 30 °C. Kanamycin-resistant colo-nies with in-frame pmrL replacements were initially confirmedby colony PCR, using external primers located 100 bp upstreamand 100 downstream of pmrL. The genomic DNA from a col-ony yielding a fragment of the correct size was subsequentlyprepared and used as the template to amplify larger amounts ofthe same fragment, using the same pair of the external primersas in the colony PCR. A single PCR product was resolved on a1% agarose gel, purified, and sequenced using the same primersto confirm the gene replacement. The resulting strain was des-ignated as AY100 (pmrL::kan) (Table 1).The same general methods were used to construct in-frame

replacements of pmrM (AY101), of pmrLM (AY102), and ofarnT (AY103) (Table 1). In each case, the same kanamycinresistance cassette was introduced into the pmrAc parentalstrain MST100. However, in the pmrM and the pmrLM dele-tion strains, no ribosomal binding site was introduced at the 3�end of the kanamycin resistance cassette, because the pmrDregulatory gene (Fig. 2B), which is immediately downstream ofpmrM, is transcribed in the opposite direction (44, 45). All con-structs were verified by colony PCR and full DNA sequencing.The sequences of the primers used for generating all of theseconstructs are provided in supplemental Table 1.Construction of Covering Plasmids—The pmrL gene of E. coli

was initially cloned into pET24b (Novagen) behind the T7lacpromoter. The coding region for pmrL was amplified by PCRfrom E. coli W3110 genomic DNA. The forward primer con-tained a clamp region, an NdeI site (supplemental Table 1,underlined), and the pmrL-coding region with its start codon(boldface). The reverse primer contained a clamp region, anXhoI site (supplemental Table 1, underlined), and the codingregion with its stop codon (boldface). Sequences of the forwardand reverse primers are shown in supplemental Table 1. ThePCR product and the pET24b vector were both digested withNdeI and XhoI. The linearized vector was treated with shrimp

TABLE 1Bacterial strains and plasmids

Description Source or Ref.StrainsE. coliW3110 Wild type, F�, �� E. coli Genetic Stock Center, Yale UniversityDY330 W3110, �lacU169 gal490 �cl857 �(cro-bioA) 47MST100 DY330, pmrAc 28SDB200 MST100, �arnD::kan, Kanr S. Breazeale, manuscript in preparationAY100 MST100, �pmrL::kan, Kanr This workAY101 MST100, �pmrM::kan, Kanr This workY102 MST100, �pmrLM::kan, Kanr This workAY103 MST100, �arnT::kan, Kanr This workNovaBlue (DE3) �(srl-recA)30::Tn10(DE3), Tetr NovagenXL-1 Blue-MR �mcrABC, recA1, lac Stratagene

PlasmidspET24b Vector containing a T7lac promoter, Kanr NovagenpET28b Vector containing a T7lac promoter, Kanr NovagenpWSK29 Low copy number vector, Ampr 48pET-PmrL pET24b expressing E. coli pmrL This workpET-PmrM pET24b expressing E. coli pmrM This workpET-PmrLM pET24b expressing E. coli pmrL & pmrM This workpET-ArnT pET21 expressing E. coli arnT 31pWSK29-PmrL pWSK29 expressing E. coli pmrL This workpWSK29-PmrM pWSK29 expressing E. coli pmrM This workpWSK29-PmrLM pWSK29 expressing E. coli pmrL and pmrM This workpWSK29-ArnT pWSK29 expressing E. coli arnT This work

TABLE 2Alternative names of gene products involved in L-Ara4N biosynthesisand transfer to lipid A

Functionalnames

Pmr terminology(S. typhimurium)a

Older designations(E. coli)b

ArnA PmrI YfbG, Orf3, b2255ArnB PmrH YfbE, Orf1, b2253ArnC PmrF YfbF, Orf2, b2254ArnD PmrJ YfbH, Orf4, b2256ArnE PmrL YfbW, Orf6, b4544ArnF PmrM YfbJ, Orf7, b2258ArnT PmrK YfbI, Orf5, b2257

a This nomenclature was used by Gunn et al. (16) for Salmonella.b This nomenclature was used in the E. coli genome project (44, 45).




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alkaline phosphatase (U. S. Biochemical Corp.) for 0.5 h at37 °C, followed by inactivation of the phosphatase at 65 °C for15 min. The vector and the PCR product were then ligatedtogether using the rapid DNA ligation kit (Roche Applied Sci-ence) and transformed into XL-1 Blue cells (Stratagene) forpropagation of the desired plasmid, designated pET-PmrL. Theinsertion was further verified by enzyme digestion and DNAsequencing. The same approach was used to construct pET-PmrM and pET-PmrLM (Table 1). The primers for construc-tion of pET-PmrM and pET-PmrLM are also shown in supple-mental Table 1.The pmrL, pmrM, and contiguous pair of pmrLM genes were

moved from the pET vector into the covering plasmid pWSK29(48), a lac-inducible, low copy expression vector useful forcomplementation experiments. The XbaI/XhoI-digested frag-ments, consisting of the either pmrL, pmrM, or the contiguouspair of pmrLM genes downstream of a pET24b-derived ribo-some-binding site, were ligated into the corresponding restric-tion sites of pWSK29. These plasmids, designated as pWSK29-PmrL, pWSK29-PmrM, or pWSK29-PmrLM (Table 1), werethen electroporated into the appropriate pmrL or pmrM dele-tion strains to test for complementation.Extraction and Separation of Phospholipids from the pmrAC

Parent MST100 or Various Mutants—Typically, 1 ml of over-night culture from a freshly streaked single colony was inocu-lated into 100 ml of LB broth (for MST100) or into LB brothcontaining 20 �g/ml kanamycin (for the various mutantstrains). The cells were grown until A600 reached �1.0. Theywere collected by centrifugation and washed with 25 ml ofphosphate-buffered saline, pH 7.4. To extract the phospholip-ids, the cell pellet was resuspended in 19 ml of a single-phaseBligh/Dyer mixture (49), consisting of chloroform/methanol/water (1:2:0.8, v/v). After mixing and incubating for 60 min atroom temperature, the insoluble debris pellet was removed bycentrifugation, and the supernatant containing the phospholip-ids was transferred to a clean glass tube. The supernatant wasconverted to a two-phase Bligh/Dyer system consisting of chlo-roform/methanol/water (2:2:1.8 v/v) by adding appropriateamounts of chloroform and water. The phases were separatedby low speed centrifugation (4000� g), and the lower phasewascollected. The samplewas dried under a streamofN2 and redis-solved in a small volume of chloroform/methanol (2:1, v/v).Typically, 20 �l of this sample was further diluted 10-fold withchloroform/methanol (1:1, v/v) for electrospray ionization-time of flight mass spectrometry (ESI/MS) analysis. The rest ofthe sample was dried under a stream of N2 andwas subjected tomild alkaline hydrolysis.Mild Alkaline Hydrolysis of Lipids—Mild alkaline hydrolysis to

deacylate the glycerophospholipids was performed as described(50). Typically, an alkaline methanol solution was prepared byadding 0.4 ml of 5 M NaOH to 5 ml of methanol (final NaOHconcentration of 0.37 M). After mixing, 5 ml of chloroform wasadded. The N2-dried phospholipids, prepared as describedabove, were redissolved in 1ml of thismixture and incubated atroom temperature for 1 h. TheNaOHwas neutralizedwith 3–4drops of concentrated HCl, as judged by pH paper, and thesystem was converted to a two-phase Bligh-Dyer mixture (49).The lower phase was retrieved and dried under a stream of N2.

The sample was then redissolved in 50 �l of chloroform/meth-anol (2:1, v/v), and 40 �l of this solution was spotted onto aSilica Gel 60 TLC plate, which was developed in the solventchloroform/methanol/water/acetic acid (25:15:4:4, v/v). Theremaining 10 �l was further diluted 10-fold with chloroform/methanol (1:1, v/v) for ESI/MS analysis.Extraction of Lipid A from the pmrAC Parental Strain

MST100 or Various Mutants—Cells were grown, harvested,and washed as described above. Each cell pellet was extractedwith 120 ml of a single phase Bligh/Dyer mixture (49). After 60min at room temperature, themixture was subjected to centrif-ugation (4,000 � g for 20 min). The resulting cell debris pelletwas extracted two more times with 120 ml of a single phaseBligh/Dyermixture. The final insoluble residue, which containslipopolysaccharide, was subjected to hydrolysis at 100 °C in 25mM sodium acetate buffer, pH 4.5, in the presence of 1% SDS tocleave the Kdo-lipid A linkage (51). The released lipid Amolec-ular specieswere extractedwith a two-phase Bligh/Dyer systemby adding appropriate amounts of chloroform and methanol.The lower phase was saved, and the upper phase was washedonce with a pre-equilibrated acidic lower phase. The pooledlower phaseswere dried under a streamofN2. The isolated lipidA was redissolved in chloroform/methanol (4:1, v/v). A portionof the samplewas subjected to ESI/MS analysis, and a portion ofthe remainderwas spotted onto a SilicaGel 60TLCplate, whichwas developed in the solvent chloroform, pyridine, 88% formicacid, water (50:50:16:5, v/v). The plate was dried, sprayed with10% sulfuric acid in ethanol, and then charred on a hot plate tovisualize the lipid A species.Sulfo-NHS-Biotin and NHS-Biotin Modification of Undeca-

prenyl Phosphate L-Ara4N in the Parent MST100 or in MutantStrains—Overnight cultures of the pmrAc parental strainMST100, and its derivatives AY100 (pmrL), AY101 (pmrM), orAY103 (arnT) were diluted 1:100 into 120 ml of LB broth, pH7.0. Cells were grown with shaking at 30 °C until the density(A600) reached 1.0. Cells were harvested by centrifugation at4,000� g for 20min andwashed once with 50ml of phosphate-buffered saline, pH 7.4 (52). For optimal labelingwith the biotinreagents, the intact cells were subsequently washed twice with50ml of modified phosphate-buffered saline, pH 7.9, as recom-mended by the manufacturer. Cells were then resuspended in12 ml of phosphate-buffered saline, pH 7.9, and divided intothree portions (no labeling control, labeled with sulfo-NHS-biotin, or labeled with NHS-biotin). Next, 40 �l of 200 mMsulfo-NHS-biotin dissolved in water or, alternatively, 40 �l of200 mMNHS-biotin dissolved inMe2SO were added to the cellsuspensions, which were incubated at 4 °C for various times.The final concentrations of the labeling reagents were 2 mM.Reactions were quenched by the addition of 25 ml of 50 mMglycine, and the cells were collected by centrifugation. The cellpellets were extracted to recover the lipids (49), which weresubjected to mild alkaline hydrolysis, as described above (50).The samples were then dissolved in the appropriate solvent forTLC or ESI/MS analysis.ESI/MS of Lipid Preparations—All mass spectra were

acquired on a QSTAR XL quadrupole time-of-flight tandemmass spectrometer (ABI/MDS-Sciex, Toronto, Canada),equipped with an electrospray ionization source. Spectra were




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acquired in the negative ion mode, and typically were the sum-mation of 60 scans from 200 to 2000 atomicmass units. ForMSanalysis, the lipid A ormild alkali stable lipid preparations were

dissolved in 200 �l of chloroform/methanol (4:1, v/v) or chlo-roform/methanol (2:1, v/v). Typically, 20�l of thismaterial wasfurther diluted into 200 �l of chloroform/methanol (1:1, v/v),containing 1% piperidine, and immediately infused into the ionsource at 5–10 �l/min. The negative ion ESI/MS was carriedout at �4200 V. In the MS/MS mode, collision-induced disso-ciation tandem mass spectra were obtained using a collisionenergy of �80 V (laboratory frame of energy). Nitrogen wasused as the collision gas. Data acquisition and analysis wereperformed using Analyst QS software.

RESULTS

Relationship of PmrL and PmrM to the Small MultidrugResistance Efflux PumpExporters—ABLASTp analysis of PmrLand PmrM shows that both are small integral membrane pro-teins with four putative trans-membrane helices. The PmrLand PmrM sequences (111 and 128 amino acid residues respec-tively) are distantly related to each other and to the drug/me-

tabolite transporter superfamily(37, 38, 53). The hydropathy plots ofthe two proteins resemble that ofEmrE, an exporter of positivelycharged hydrophobic drugs inE. coli (54). X-ray structures ofEmrE with or without the boundsubstrate tetraphenylphosphoniumwere reported by Chang andco-workers (55, 56) but wererecently retracted (57). Orthologuesof PmrL and PmrM exist in manybacteria, but their functions arepoorly characterized. Orthologueswith strong sequence similarity toPmrL and PmrM are present in allstrains of E. coli, Salmonella,Yersinia pestis, Yersinia pseudotu-berculosis, and Pseudomonasaeruginosa that modify their lipid Awith the L-Ara4N moiety (2, 58).Given their sequences and genomiccontext (Fig. 2B), PmrL and PmrMmight function as part of an innermembrane transporter or flippasesystem for undecaprenyl phos-phate-�-L-Ara4N (Fig. 2A).Deletion Mutants of pmrL and

pmrM in MST100 Regain Poly-myxin Sensitivity—To investigatethe roles of the contiguous pmrLand pmrM genes in the polymyxinoperon (Fig. 2B), we generatedchromosomal deletions of pmrLand/or pmrM in the polymyxin-re-sistant strain MST100 (pmrAc),using in-frame kanamycin cassettereplacements. In the case of thepmrL knock-out, a ribosome-bind-ing site was inserted between the

FIGURE 3. The �pmrL::kan and �pmrM::kan mutations confer polymyxinsensitivity on the pmrAc strain MST100. The in-frame replacements of pmrLand pmrM with the kan cassette were constructed as described under “Exper-imental Procedures.” Strains were streaked on LB agar or on LB agar contain-ing either 15 �g/ml polymyxin or 20 �g/ml kanamycin. Cells were then grownovernight at 30 °C. Numbers indicate the following strains: 1, MST100 (pmrAc);2, SDB200 (pmrAc �arnD::kan); 3, AY100 (pmrAc �pmrL::kan); 4, AY101 (pmrAc

�pmrM::kan); and 5, AY102 (pmrAc �pmrLM::kan).

FIGURE 4. Loss of L-Ara4N-modified lipid A species in �pmrL::kan and �pmrM::kan mutants of pmrAc strainMST100. Lipid A was isolated from the strains indicated and was subjected to ESI/MS analysis in the negative ionmode. Compared with the parental strain MST100, shown in A, the �pmrL and �pmrM strains (B and C, respectively)lacked a series of L-Ara4N modified lipid A species (red numbers in A). The proposed compositions of the numberedpeaks and their expected exact masses are listed in Table 3. amu, atomic mass units.




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stop codon of kan gene and the start codon of the downstreampmrM gene to ensure proper expression of PmrM. In thepmrM-deleted construct, an extra sequence of nucleotides cor-responding to the overlapping region between pmrM and thedownstream regulatory pmrD gene (Fig. 2B), which is tran-scribed in the opposite direction (45), was inserted behind thestop codon of the kan gene in the primer (supplemental Table1) to avoid altered regulation of pmrD expression.We also con-structed a kan replacement of both the pmrL and pmrM genesin MST100. These isogenic mutants and the parental strainwere then tested for polymyxin resistance. An arnD deletionmutant (Fig. 2,A andB) ofMST100, previously shown to regainpolymyxin sensitivity, was utilized as another control.5 Asshown in Fig. 3, the pmrL and the pmrM deletion mutants (aswell as the double deletion mutant) were sensitive to 15 �g/mlpolymyxin, indicating that both genes participate in the main-tenance of polymyxin resistance in E. coli. The growth of themutants was not impaired on LB agar in the absence of poly-myxin, and both were resistant to kanamycin (Fig. 3).Polymyxin resistance was specifically restored to the pmrL

deletionmutantAY100 by transformationwith pWSK29-PmrL(Table 1) but not with pWSK29-PmrM (data not shown). Con-versely, polymyxin resistance was recovered in the pmrM dele-tion mutant AY101 by transforming with pWSK29-PmrM(Table 1), but not with pWSK29-PmrL (data not shown), dem-onstrating that both pmrL and pmrM play indispensable rolesin the maintenance of polymyxin resistance in MST100.The pmrL and pmrM Deletion Mutants Lack L-Ara4N-mod-

ified Lipid A Species—Because the L-Ara4N modification oflipid A is required for polymyxin resistance (2), the status oflipid Amodification with the L-Ara4Nmoiety was examined ineach of the mutants. Crude lipid A, obtained by pH 4.5 hydrol-ysis of a chloroform/methanol-extracted cell residue, was sub-jected to TLC (data not shown) and ESI/MS analysis (Fig. 4). Asignificant fraction of the lipid A molecular species present inthe parental strain MST100 was modified with the L-Ara4Nunit (Fig. 4, peaks with red numbers, and Table 3). In addition,lipid A molecules bearing one or two pEtN units (but noL-Ara4N moiety) were also present in the parental strain (Fig.4A, peaks with black numbers, and Table 3). In the pmrL or

pmrM deletionmutants, over 95%of each of the L-Ara4N-mod-ified lipid A species was missing (Fig. 4, B and C). In contrast,the pEtN and/or palmitate-modified lipid A species (Figs. 1 and4 andTable 3)were present at normal or elevated levels. As seenpreviously with S. typhimurium arnC (pmrF) mutants (Fig. 2),the relative amounts of lipid A species bearing two pEtN units(Fig. 4, peaks 5 and 7) were actually elevated in the mutantsunable to add the L-Ara4N unit to lipid A (21). These resultsimply that the PmrL and PmrM proteins are involved in thebiosynthesis, trafficking, or attachment of the L-Ara4N unit tolipid A (Fig. 2).5 S. D. Breazeale and C. R. H. Raetz, manuscript in preparation.

FIGURE 5. Undecaprenyl phosphate-�-L-Ara4N levels in pmrAc strainMST100 and its �pmrL::kan and �pmrM::kan derivatives. The pmrA con-stitutive parental strain, MST100, as well as the derived mutants AY100(�pmrL), AY101 (�pmrM), and AY102 (�pmrLM) were grown to mid-logphase. The lipids were extracted and subjected to mild alkaline hydrolysis, asdescribed under “Experimental Procedures.” The hydrolysis products wereseparated by silica TLC, using the solvent chloroform/methanol/acetic acid/H2O (25:15:4:4, v/v), and visualized by charring on a hot plate after sprayingwith 10% sulfuric acid in ethanol.

TABLE 3Exact masses of modified lipid A species and assignments of the major peaksObserved masses are taken from Fig. 4A. In the pmrL and pmrM deletion mutants (Fig. 4, B and C) an additional peak (not shown) atm/z 1150.7 is attributed to a lipid Aspecies modified with two pEtN and one palmitoyl substituent.

Covalent group(s) Peak no. Exact mass Predicted �M � 2H�2� Observed �M � 2H�2� Predicted �M � Na-3H�2� Observed �M � Na-3H�2�

L-Ara4N 2 1928.278 963.131 963.114L-Ara4N 4 1928.278 974.122 974.106L-Ara4N 6 2051.286 1024.635 1024.619pEtNL-Ara4N 8 2051.286 1035.626 1035.612pEtNL-Ara4N 9 2166.507 1082.246 1082.228C16:0pEtN 1 1920.228 959.106 959.089pEtN 3 1920.228 970.097 970.078(pEtN)2 5 2043.236 1020.610 1020.593(pEtN)2 7 2043.236 1031.601 1031.580pEtN 10 2158.458 1089.212 1089.188C16:0




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Undecaprenyl Phosphate-�-L-Ara4N Levels in pmrL andpmrMMutants of MST100—The expression of the other geneproducts in the polymyxin resistance operon (Fig. 2, A and B)should not be affected by the pmrL or pmrM deletions. Accord-ingly, the intermediates of the pathway (Fig. 2A) should be pres-ent in normal amounts. Given that the modification of lipid Awith L-Ara4N was largely abolished upon disruption of eitherthe pmrL or the pmrM gene, it was conceivable that undecapre-nyl phosphate-�-L-Ara4Nmight actually accumulate to higherlevels than seen in the parental strain. We therefore extractedthe phospholipids from the parent andmutant strains and sub-jected them to mild alkaline hydrolysis to remove the glycero-phospholipids. TLC (Fig. 5) and ESI/MS analysis (see below)demonstrated that undecaprenyl phosphate-�-L-Ara4N levelswere the same or slightly higher in the mutants than in theparent, indicating that appropriate amounts of the prenol lipiddonor of the L-Ara4N moiety are still synthesized in thesemutants.The active site of ArnT, which transfers the L-Ara4Nmoiety

from undecaprenyl phosphate-�-L-Ara4N to lipid A, is thoughtto be located on the periplasmic surface of the innermembrane(13, 31). The failure to attach the L-Ara4N moiety to lipid A inour mutants is therefore consistent with a defect in the trans-location of undecaprenyl phosphate-�-L-Ara4N from its site ofbiosynthesis on the inner surface of the inner membrane tothe outer surface of the inner membrane (Fig. 2A).Reduced Labeling of Undecaprenyl Phosphate-�-L-Ara4N by

Sulfo-NHS-Biotin in pmrL and pmrM Mutants—To evaluatethe roles of PmrL and PmrM in undecaprenyl phosphate-�-L-Ara4N transport across the inner membrane, cells weretreated with the inner membrane-impermeable amine reagent,

sulfo-NHS-biotin (Scheme 1). Sulfo-NHS-biotin is a hydro-philic, low molecular weight biotinylation reagent that reactswith any primary amine. It is able to diffuse through the outermembrane porins of E. coli to reach the periplasmic space (36),but it cannot enter the cytoplasm because it does not cross thephospholipid bilayer of the inner membrane (40). Accordingly,if undecaprenyl phosphate-�-L-Ara4N were trapped on theinner surface of the inner membrane in the PmrL- and/orPmrM-deficient strains, sulfo-NHS-biotin labeling of theendogenous undecaprenyl phosphate-�-L-Ara4N should bereduced, when compared with the parental strain or to a com-parable arnT (Fig. 2) deletion mutant.

To evaluate undecaprenyl phosphate-�-L-Ara4N accessibil-ity, we labeled MST100 and the mutant cells for various timeswith sulfo-NHS-biotin. We then analyzed the extent of unde-caprenyl phosphate-�-L-Ara4Nmodification by TLC (Fig. 6) orESI/MS (Fig. 7). As shown in Fig. 6A for MST100, a new com-pound, proposed to be biotinylated undecaprenyl phosphate-�-L-Ara4N (Scheme 1), was generated over the course of 4 h.This correlated with the appearance of a singly charged peak atamass-to-charge ratio (m/z) of 1202.785 in the negative ion ESImass spectrum (Fig. 7,Aversus B), consistentwith the structureshown in Scheme 1. Themonoisotopic peak area ratio of unmod-ifiedundecaprenyl phosphate-�-L-Ara4N (m/z976.707) to biotin-ylated undecaprenyl phosphate-�-L-Ara4N (m/z 1202.785) was0.069 (Fig. 7B) at this time point (0.001 for technical repli-cates). The actual molar ratio may be higher, as the ioniza-tion efficiency of the biotinylated undecaprenyl phosphate-�-L-Ara4N (Scheme 1) is likely to be somewhat lower thanthat of the unmodified material under the electrospray con-ditions employed.In the pmrL and pmrM mutants, significantly less biotiny-

lated undecaprenyl phosphate-�-L-Ara4N was generated, asjudged by both TLC (Fig. 6, B and C) and ESI/MS (Fig. 7, C andD). At the 4-h time point, there was a 4–5-fold reduction inratio of the monoisotopic peak areas for biotinylated undeca-prenyl phosphate-�-L-Ara4N compared with unmodifiedundecaprenyl phosphate-�-L-Ara4N (Fig. 7, C and D), despitecomparable levels of undecaprenyl phosphate-�-L-Ara4N in allstrains (Fig. 5 and Fig. 7, B versus C and D). A liquid chroma-tography ESI/MS analysis of all the samples gave similar results(data not shown). The data imply that bothPmrL andPmrMareinvolved in the translocation of undecaprenyl phosphate-�-L-Ara4N across the inner membrane.Labeling of Undecaprenyl Phosphate-�-L-Ara4N by the

Membrane-permeable Reagent NHS-Biotin—To exclude otherpossibilities, such as sequestration by protein binding, for thereduced modification of undecaprenyl phosphate-�-L-Ara4Nby sulfo-NHS-biotin in pmrL and pmrMmutants, we examinedundecaprenyl phosphate-�-L-Ara4N reactivity toward thehydrophobic amine reagent, NHS-biotin, under otherwiseidentical conditions. This compound is able to cross both theouter and the inner membrane of E. coli, and it should labelundecaprenyl phosphate-�-L-Ara4N with equal efficiency,irrespective of its membrane localization. Use of NHS-biotinshould generate similar amounts of biotinylated undecapre-nyl phosphate-�-L-Ara4N in the parental and mutantstrains. Similar levels of biotinylated undecaprenyl phos-

SCHEME 1. Biotinylation of undecaprenyl phosphate-�-L-Ara4N by sulfo-NHS-biotin or NHS-biotin. The same lipid product is generated with bothreagents.




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phate-�-L-Ara4N were indeed observed in all the strainswith NHS-biotin, as demonstrated by TLC (Fig. 8, lanes 1–3)and ESI/MS analysis (not shown), although the rate of aminegroup labeling with NHS-biotin was slightly slower thanwith sulfo-NHS-biotin.ArnT Is Not Involved in the Transport of Undecaprenyl

Phosphate-�-L-Ara4N—Given its 12 predicted trans-mem-brane helices (31), the enzymeArnT (Fig. 2)might function notonly to attach the L-Ara4Nunit to lipidA but also to translocateundecaprenyl phosphate-�-L-Ara4N across the inner mem-brane. Accordingly, we constructed an in-frame kan replace-ment of the arnT gene, using the same MST100 backgroundstrain as for the pmrL and pmrM deletions. Lipid A modifica-tion with L-Ara4N was abolished in the arnT knock-out (datanot shown), as in the pmrL and pmrM deletions (Fig. 4, B andC). The high levels of undecaprenyl phosphate-�-L-Ara4N seenin MST100 were unaffected by the disruption of the arnT gene(Fig. 6, A versus D, and Fig. 7, B versus E). In contrast to thepmrL and pmrM mutants, however, the undecaprenyl phos-phate-�-L-Ara4N present in the arnTmutant reacted with thehydrophilic reagent sulfo-NHS-biotin exactly as it did in theparental strain (Fig. 7, B versus E), yielding a peak area ratio of0.068 (Fig. 7E). These results are consistent with the idea thatthe undecaprenyl phosphate-�-L-Ara4N gains access to theperiplasmic side of the inner membrane independently of arnTfunction. When the arnT mutant cells were labeled with thehydrophobic reagent NHS-biotin, modified undecaprenylphosphate-�-L-Ara4N (Fig. 8, lane 4) was formed as effi-

ciently as in MST100 (Fig. 8, lane 1) and in the pmrL orpmrM deletions (Fig. 8, lanes 2 and 3). These data suggestthat ArnT does not participate in translocating its donorsubstrate, undecaprenyl phosphate-�-L-Ara4N, to its activesite. Instead, PmrL and PmrM could specifically function toflip undecaprenyl phosphate-�-L-Ara4N from the cytosolicto the periplasmic side of the inner membrane, possiblyfunctioning as a heterodimer. Given their likely roles astransporters in the L-Ara4N pathway, we suggest that PmrLand PmrM be renamed ArnE and ArnF (Fig. 2B).

DISCUSSION

Phospholipid flip-flop across biological membranes is animportant yet poorly characterized process. The slow rate ofphospholipid flip-flop in model lipid bilayers compared withbiological membranes first suggested that specific proteinsmight be required to facilitate this process (59–62). The bestcharacterized lipid transporters aremembers of the ATP-bind-ing cassette (ABC) transporter superfamily (63, 64), whichhydrolyze ATP to drive the unidirectional transport of specificcompounds, including many lipids, across biological mem-branes. For instance, mouse mutants lacking the ABC trans-porter Mdr2 lack phosphatidylcholine in their bile (65).Patients with sitosterolemia lack the ABC transporters thatpump absorbed sterols, including the plant-derived sitosterol,back into the gut or the bile (66). In bacteria, point mutants inthe essential ABC transporter MsbA are defective in flippingnascent LPS across the inner membranes at elevated tempera-tures (35, 36, 67, 68), as judged by accessibility to periplasmiclipid A modification enzymes. Such mutants are also defectivein glycerophospholipid export to the outer membrane (35), butthis phenomenon might be secondary to LPS accumulationwithin the inner membrane. Despite the compelling geneticand physiological evidence for their roles in lipid trafficking,no simple, generally applicable, assays have been devised fordemonstrating ABC transporter-dependent flipping of specificlipids in vitro.Bacteria possess other types of transporters that use the

energy from proton or electrochemical gradients to pumpchemicals or ions cross the cytoplasmic membrane (37, 38, 53).For instance, the small multidrug resistance transporters aretypically 105–121 amino acids long, contain four predictedtrans-membrane helices, and function as homo- or hetero-oligomers (37, 38, 53, 54). Examples include heterodimericcomplexes, such as EbrA/EbrB of Bacillus subtilis (39), as wellas the homodimeric transporter EmrE of E. coli (54). The EmrEtransporter confers resistance to lipophilic cations, such as tet-raphenylphosphonium, methyl viologen, and ethidium (69), aswell as to certain antibiotics (70). Although a published crystalstructure of EmrE (55, 56) was recently retracted (57), and itsproposed mechanism (55, 56) must be therefore questioned,there is no doubt that EmrE functions to export lipophilic cat-ions from living bacteria (71). In vitro transport assays depend-ent upon purified reconstituted EmrE have been described (72).PmrL and PmrM are small, hydrophobic, inner membrane

proteins with many of the characteristics of small multidrugresistance transporters. Both PmrL and PmrM are predicted tohave four membrane-spanning helices. These proteins are the

FIGURE 6. Reduced labeling of undecaprenyl phosphate-�-L-Ara4N bysulfo-NHS-biotin in PmrL and PmrM mutants of MST100. The pmrAconstitutive parental strain MST100 (A), as well as the derived mutantsAY100 (�pmrL) (B), AY101 (�pmrM) (C), and AY103 (�arnT) (D) were grown tomid-log phase. The washed cells were treated with sulfo-NHS-biotin at a finalconcentration of 2 mM for 0, 1, 2, or 4 h at 4 °C. Reactions were quenched with50 mM glycine. Phospholipids were extracted and were subjected to mildbase hydrolysis (50). The base-stable prenol lipids were separated by TLC,using the solvent chloroform/methanol/acetic acid/H2O (25:15:4:4, v/v), andvisualized by charring as in Fig. 5.




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distal genes of an operon requiredfor the maintenance of polymyxinresistance (16) (Fig. 2B), which ispresent in both E. coli and Salmo-nella, and is regulated by the PmrA-(BasR) transcription factor (73), inconjunction with the PmrB andPmrD proteins (19, 74). We havenow demonstrated that in-framechromosomal deletions of pmrL,pmrM, or both restore polymyxinsensitivity to a PmrA-constitutive,polymyxin-resistant parental strain(Fig. 3). Concomitantly, the L-Ara4N-modified lipid A species (Fig. 4) areno longer synthesized in largeamounts. However, careful inspec-tion of the spectrum (Fig. 4,B andC)suggests that small amounts ofL-Ara4N-modified lipid A speciesare still present, whereas they arecompletely absent in the ArnT orArnA deletion mutants (data notshown). ThepmrL andpmrMmuta-tions do not inhibit the biosynthesisof undecaprenyl phosphate-�-L-Ara4N (Fig. 5), which is the imme-diate donor of the L-Ara4N unit tolipid A. However, the apparent trans-location of undecaprenyl phosphate-�-L-Ara4N from the cytoplasmic tothe periplasmic side of the innermembrane is reduced by 4–5-fold, asjudged by accessibility to labelingwith the impermeable reagent sulfo-NHS-biotin (Figs. 6 and 7). We pro-pose that PmrL and PmrM berenamed ArnE and ArnF, respec-tively (Fig. 2B), given their functionin generating L-Ara4N modifiedlipid A species (Fig. 4).It is possible that ArnE and ArnF,

like some of the multidrug effluxpumps (39), form a heterodimer invivo. The systematic studies by vonHeijne and co-workers (75–77) onthe topology of E. coli membraneproteins are consistent with theArnE/ArnF model shown in Fig. 9.This proposal could be tested fur-ther by immunoprecipitation orpurification of ArnE and/or ArnF.ArnE and ArnF share low sequencesimilarity to each other and toE. coliEmrE. All three proteins displaysimilar overall hydropathy profiles.It will also be important to deter-mine whether or not metabolic




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energy is required for the functioning of ArnE/F, for instance,the proton motive force across the inner membrane or theavailability of ATP.The origin of the �20% residual sulfo-NHS-biotin-labeled

material in the prmL- and pmrM-deficient strains (Fig. 7,C andD) is uncertain. It may be that under our conditions of labelingsome sulfo-NHS-biotin does reach the cytoplasm or some ofthe cells have become leaky. Alternatively, there may be othermembrane proteins that can flip undecaprenyl phosphate-�-L-Ara4N across the inner membrane at a slower rate.Membrane-impermeable reagents, such as sulfo-NHS-biotin

or 2,4,6-trinitrobenzene sulfonic acid, have previously been uti-lized to characterize phosphatidylethanolamine flip-flop inwhole cells (36, 40). We have successfully utilized sulfo-NHS-biotin (Figs. 6 and 7) and its membrane-permeable analogueNHS-biotin (Fig. 8) to measure the accessibility of undecapre-nyl phosphate-�-L-Ara4N to covalent modification. Negativeionmode ESI/MSwas used for the first time as an unambiguouscriterion of undecaprenyl phosphate-�-L-Ara4N modification.Our results clearly show that ESI/MS is an efficient and defini-tive tool for monitoring lipidmodification withmembrane-im-permeable reagents. It may be possible to adapt this approach

to assay the flip-flop of other amine-containing lipids in wholecells or in isolated membrane vesicles.Many other biogenic processes employ polyisoprene phos-

phate-oligosaccharides as precursors for extracellular glycosy-lation reactions. These systems includeN-linked protein glyco-sylation in eukaryotic (78–80) and bacterial cells (81, 82), thegeneration of LPS O-antigen (1), the assembly of enterobacte-rial common antigen (83), and the polymerization of pepti-doglycan (84). Although the sugar compositions and linkages ofthese polyisoprene phosphate-oligosaccharides differ greatly,two commonprinciples emerge. First, a polyisoprene derivative(dolichyl phosphate in eukaryotic cells or undecaprenyl phos-phate inE. coli) functions as the acceptor for one ormore sugarsresidues, which are donated by cytoplasmic sugar nucleotides.Second, the polyisoprene phosphate-sugar intermediates aretranslocated (flipped) to the lumen of the endoplasmic reticu-lum in eukaryotic cells or to the periplasmic surface of the innermembrane in Gram-negative bacteria.Recently, several specific membrane proteins have been

implicated as polyisoprene phosphate-oligosaccharide flip-pases in yeast and bacteria. The yeast membrane protein Rft1pis proposed to flip dolichyl diphosphate-GlcNAc2Man5 fromthe cytoplasmic to the luminal side of the endoplasmic reticu-lum (78). The ABC transporter PglK (WlaB) in the humanpathogen Campylobacter jejuni translocates an undecaprenyl-diphosphate-linked heptasaccharide from the cytoplasmic tothe periplasmic surface of the inner membrane (82). The Wzxproteins of Gram-negative bacteria appear to translocateO-an-tigen subunits attached to undecaprenyl-diphosphate acrossthe inner membrane (85, 86) prior to polymerization andattachment to core-lipid A. None of these transporters arerelated in sequence to ArnE or ArnF. The flippases for the pep-tidoglycan precursors remain unknown. Generally applicablein vitro assays for these polyisoprene phosphate-sugar flippaseshave not been reported, although the use of short chain iso-prenes is a very promising approach in some instances (87).Although our genetic and biochemical studies implicate

FIGURE 7. ESI/MS of undecaprenyl phosphate-�-L-Ara4N and its sulfo-NHS-biotin-modified derivative in parental and mutant strains. The pmrAconstitutive parent MST100 and the mutants AY100, AY101, and AY103 were grown to mid-log phase. The washed cells were treated with sulfo-NHS-biotin at2 mM for 4 h at 4 °C. Reactions were quenched with 50 mM glycine. Lipids were extracted and subjected to mild base hydrolysis (50). The base-stable prenollipids were analyzed by negative ion ESI/MS. A, MST100 lipids without sulfo-NHS-biotin treatment; B, MST100 lipids with sulfo-NHS-biotin for 4 h; C, AY100 lipidswith sulfo-NHS-biotin for 4 h; D, AY101 lipids with sulfo-NHS-biotin for 4 h; and E, AY103 lipids with sulfo-NHS-biotin for 4 h. The insets highlight the peaksattributed to the biotinylated derivative of undecaprenyl phosphate-�-L-Ara4N, with the MST100 parental spectrum in blue and the indicated control ormutant spectra overlaid in red. The peak area ratios are derived from the monoisotopic peak area of the derivatized lipid near m/z 1202.79 divided by themonoisotopic peak area of the underivatized lipid near m/z 976.71 for each lipid sample (see Scheme 1). amu, atomic mass units.

FIGURE 8. Modification of undecaprenyl phosphate-�-L-Ara4N by mem-brane-permeable NHS-biotin in parental and mutant strains. The pmrAconstitutive parental strain MST100 (1), as well as AY100 (�pmrL) (2),AY101(�pmrM) (3), and AY103 (�arnT) (4) were grown to mid-log phase. Thewashed cells were treated with NHS-biotin at 2 mM for 4 h at 4 °C. Reactionswere quenched with 50 mM glycine. Lipids were extracted and subjected tomild alkaline hydrolysis (50). The hydrolysis products were separated by TLC,using the solvent chloroform/methanol/acetic acid/H2O (25:15:4:2, v/v), andvisualized by charring as in Fig. 5.

FIGURE 9. Proposed topography of the ArnE/ArnF heterodimer. Thismodel is based on the systematic studies of E. coli membrane proteins by vonHeijne and co-workers (75–77, 95).




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ArnE and ArnF as flippases for undecaprenyl phosphate-�-L-Ara4N, many questions remain. For example, the amount ofundecaprenyl phosphate-�-L-Ara4N in arnE, arnF, and arnTdeletion mutants is about the same as in the parental strain.Regulatory mechanisms may limit undecaprenyl phosphate-�-L-Ara4N accumulation when it is not being used for lipid Amodification. Alternatively, there may be other pathways thatconsume undecaprenyl phosphate-�-L-Ara4N. Additionalgenetic and biochemical studies will be required to addressthese questions. Furthermore, the mechanism of ArnE andArnF action as transporters remains to be characterized with invitro assays, using purified, reconstituted preparations. A crys-tal structure of ArnE/ArnF with bound undecaprenyl phos-phate-�-L-Ara4N would be especially informative. Despite thetechnical problems encountered recently with the EmrE crystalstructure (71), we are optimistic that the high resolution struc-ture of ArnE/ArnF can be solved and will provide molecularinsights into its function as the undecaprenyl phosphate-�-L-Ara4N flippase.

Acknowledgments—We are grateful to all members of the Raetz lab-oratory for their help with experimental techniques, inspiring discus-sion, and friendship. The mass spectrometry facility in the Depart-ment of Biochemistry, Duke University Medical Center, wassupported by the LIPID MAPS Large Scale Collaborative GrantGM-069338 from the National Institutes of Health.

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Aixin Yan, Ziqiang Guan and Christian R. H. RaetzEscherichia coliResistance in

An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin

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