Extracellular Loops of Lipid A 3-O-Deacylase PagL Are ...in which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipid A 3-O deacylation

JOURNAL OF BACTERIOLOGY, Aug. 2008, p. 5597–5606 Vol. 190, No. 160021-9193/08/$08.00�0 doi:10.1128/JB.00587-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Extracellular Loops of Lipid A 3-O-Deacylase PagL Are Involved inRecognition of Aminoarabinose-Based Membrane Modifications

in Salmonella enterica Serovar Typhimurium�

Takayuki Manabe and Kiyoshi Kawasaki*Faculty of Pharmaceutical Sciences, Doshisha Women’s College, Kyotanabe, Kyoto 610-0395, Japan

Received 29 April 2008/Accepted 12 June 2008

Salmonella enterica serovar Typhimurium modifies its lipopolysaccharide (LPS), including the lipid Aportion, in response to changes in its environment including host tissues. The lipid A 3-O-deacylase PagL, theexpression of which is promoted under a host-mimetic environment, exhibits latency in S. enterica; deacylationof lipid A is not usually observed in vivo, despite the expression of the outer membrane protein PagL. Incontrast, PagL does not exhibit latency in S. enterica pmrA and pmrE mutants, both of which are deficient inthe aminoarabinose-based modification of lipid A, indicating that aminoarabinose-modified LPS species wereinvolved in the latency. In order to analyze the machinery for PagL’s repression, we generated PagL mutantsin which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipidA 3-O deacylation was observed in S. enterica expressing the recombinant mutants PagL(R43A), PagL(R44A),PagL(C85A), and PagL(R135A), but not in S. enterica expressing wild-type PagL, suggesting that the pointmutations released PagL from the latency. In addition, mutations at Arg-43, Arg-44, Cys-85, and Arg-135 didnot affect lipid A 3-O-deacylase activity in an S. enterica pmrA mutant or in Escherichia coli BL21(DE3). Theseresults, taken together, indicate that specific amino acid residues located at extracellular loops of PagL areinvolved in the recognition of aminoarabinose-modified LPS. Furthermore, S. enterica expressing the recom-binant PagL(R43A) or PagL(R135A) mutant showed apparent growth arrest at 43°C compared with S. entericaexpressing wild-type PagL, indicating that the latency of PagL is important for bacterial growth.

Exclusive areas of the outer leaflet of the outer membraneare occupied by lipopolysaccharide (LPS) molecules in gram-negative bacteria (reviewed in reference 33). LPS consists of ahydrophobic membrane anchor portion known as lipid A anda nonrepeating core oligosaccharide coupled to a distal poly-saccharide (O antigen) that extends from the bacterial surface(reviewed in reference 37). The outer membrane of pathogenicgram-negative bacteria, including Salmonella enterica serovarTyphimurium, functions as a barrier to harmful host-derivedcompounds, such as antimicrobial peptides and reactive oxy-gen. The protection is a consequence of strong lateral interac-tion between LPS molecules (reviewed in reference 33). Inresponse to environmental signals, including those from thehost, S. enterica covalently modifies its lipid A through palmi-toylation, deacylation, formation of a 2-hydroxymyristategroup (hydroxylation), and addition of 4-amino-4-deoxy-L-arab-inose (aminoarabinose) or phosphoethanolamine (Fig. 1) (re-viewed in references 9, 36, and 37). These modifications areimplicated in the virulence of pathogenic gram-negative bac-teria in that they increase resistance to cationic antimicrobialpeptides (17, 29, 34). On the other hand, the lipid A portion ofLPS, which is known as endotoxin, induces inflammatory re-sponses through recognition by Toll-like receptor 4 (TLR4)–MD2 complex (reviewed in reference 32). The modifications oflipid A in S. enterica were implicated in a reduction of recog-

nition through the TLR4-MD2 complex (26, 44), leading to theevasion of host immune responses.

A two-component regulatory system, PhoP-PhoQ, which isessential for the pathogenesis of S. enterica (11, 16, 31), pro-motes the expression of genes involved in lipid A modifications(20). PhoQ is a sensor histidine kinase that responds to envi-ronmental conditions, including those within mammalian tis-sues, which are mimicked by magnesium-limited growth me-dium (11, 12, 16, 31). In response to specific environmentalfactors, such as magnesium limitation (12), low pH (3, 35), andantimicrobial peptides (1, 2), PhoQ phosphorylates PhoP,leading to the activation of pagL and pagP encoding lipid A3-O-deacylase and lipid A palmitoyltransferase, respectively (4,6, 21, 45). Since 3-O deacylation decreases the endotoxic ac-tivity of lipid A, the modification is thought to help S. entericaevade immunosurveillance (26). Activation of PhoP-PhoQleads to the activation of a second two-component regulatorysystem, PmrA-PmrB (18, 24), which promotes the expressionof genes involved in the attachment of aminoarabinose andphosphoethanolamine to phosphate groups on lipid A (Fig. 1).The modification with aminoarabinose is essential for PhoP-PhoQ-dependent resistance to cationic antimicrobial peptides,including polymyxin B (17, 18, 29, 34).

In addition to gene expression, the posttranslational regula-tion of outer membrane enzymes, including lipid A 3-O-deac-ylase PagL, is suggested to be involved in the regulation of lipidA’s modifications. Previous studies found that lipid A was notdeacylated despite the induction of PagL expression in S. en-terica (27, 45); therefore, PagL is thought to be latent in theouter membrane (27). In contrast, PagL-dependent deacyla-tion of lipid A was detected in pmrA, pmrE, and pmrF mutants

* Corresponding author. Mailing address: Faculty of PharmaceuticalSciences, Doshisha Women’s College, Kodo, Kyotanabe, Kyoto 610-0395, Japan. Phone: 81-774-65-8588. Fax: 81-774-65-8585. E-mail:[email protected].

� Published ahead of print on 20 June 2008.

5597

on February 16, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

deficient in the aminoarabinose-based modification of lipid A(27). In addition, the release of PagL from latency in the S.enterica pmrA mutant partly compensated for susceptibility topolymyxin B, which was caused by a lack of modification oflipid A (25). These results, taken together, suggest that ami-noarabinose-containing outer membranes directly inactivatethe lipid A 3-O-deacylase activity of PagL. Alternatively, mod-ification of lipid A with aminoarabinose inhibits the physicalinteraction of LPS with PagL.

In this study, we generated PagL mutants in which an aminoacid residue located at four extracellular loops was replacedwith alanine and examined the effect on the latency. Severalmutations apparently released PagL from its latency, indicatingthe extracellular loops are involved in the recognition of ami-noarabinose-modified lipid A. Furthermore, S. enterica ex-pressing PagL mutants that lost the latency exhibited growth

arrest at 43°C, implying the physiological importance of thelatency for cell growth under specific conditions.

MATERIALS AND METHODS

Materials. All chemicals were of reagent grade or better. Restriction endo-nucleases and DNA-modifying enzymes were from New England Biolabs (Bev-erly, MA) and Takara Bio (Ohtsu, Japan). Oligonucleotides were preparedcommercially by Greiner Japan (Tokyo, Japan). Prestained molecular weightstandards were from Apro Science (Naruto, Japan). 2, 5-Dihydroxybenzoic acidwas from Sigma-Aldrich (St. Louis, MO), and 5-chloro-2-mercaptobenzothiazolewas from Wako Chemicals (Osaka, Japan). Proteinase K was from Roche Di-agnosis (Basel, Switzerland).

Bacterial strains and growth conditions. CS283 (14028s phoN2 zxx::6251Tn10d-Cam pagL1::TnphoA) (27, 31) and KCS040 (14028s phoN2 zxx::6251Tn10d-Cam pagL1::TnphoA pmrA::Tn10d) (27), which are derivatives of S. en-terica serovar Typhimurium strain 14028s (American Type Culture Collection,Manassas, VA), were used in this study. Unless otherwise indicated, S. entericacells were grown at 37°C with aeration in N-minimal medium [5 mM KCl, 7.5

FIG. 1. PhoP-PhoQ-regulated lipid A modifications in S. enterica. (A) Prototype lipid A of S. enterica serovar Typhimurium. (B) Modified lipidA of S. enterica serovar Typhimurium. The phosphate residues and acyl chains of lipid A of S. enterica can be derivatized in a PhoP-PhoQ- orPmrA-PmrB-regulated manner (reviewed in reference 9). Aminoarabinose and/or phosphoethanolamine groups can be attached to phosphateresidues, under the control of PmrA-PmrB (20, 49). Minor species were present in which the locations of the aminoarabinose and phosphoeth-anolamine groups were reversed or in which both phosphates were modified with the same substituent (49). Both the pmrF operon and pmrE arenecessary for the PmrA-PmrB-regulated attachment of aminoarabinose to lipid A (17, 49). The pmrC gene mediates the PmrA-PmrB-regulatedattachment of phosphoethanolamine to lipid A (29). The addition of the palmitate chain is catalyzed by PagP (6, 21), the formation of the2-hydroxymyristate group requires LpxO (15), and the deacylation at position 3 of lipid A is catalyzed by PagL (45). The pagL and pagP genes areregulated by PhoP-PhoQ (4), and the lpxO gene is partly regulated by PhoP-PhoQ (14, 15). PhoP-PhoQ also activates PmrA-PmrB; therefore, theaminoarabinose and phosphoethanolamine modifications occur under PhoP-PhoQ-activating conditions (20, 48).

5598 MANABE AND KAWASAKI J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 0.28% glycerol (vol/vol), 0.1%(wt/vol) Casamino Acids, 0.2 mg of thiamine/liter, and 0.1 M Tris-HCl (pH 7.4)]supplemented with 10 �M MgCl2. Ampicillin (10 �g/ml) was used for thecultivation of strains transformed with the low-copy vector pWKS30 (46) or itsderivatives. S. enterica colonies were picked out and grown overnight at 37°C in1 to 10 ml of growth medium. Unless otherwise indicated, the overnight cultureswere diluted 1:10 with fresh growth medium and then grown at 37°C for 12 h.After the cultivation, stationary-phase cells were used for further analysis.

E. coli BL21(DE3) (Invitrogen) cells were grown at 37°C with aeration in LBmedium. Ampicillin (100 �g/ml) was used for the cultivation of strains trans-formed with pBluescript II KS(�) (Stratagene, La Jolla, CA) or its derivatives.E. coli colonies were picked out and grown overnight at 37°C in 3 ml of growthmedium. The overnight cultures were diluted to an optical density at 600 nm of0.1 and then grown at 37°C for 16 h. After the cultivation, stationary-phase cellswere used for further analysis.

Bacterial genetic and molecular biology techniques. Plasmid DNA was intro-duced into bacterial strains by electroporation using a Gene Pulser (Bio-Rad,Hercules, CA) following the manufacturer’s instructions. Recombinant DNAtechniques were performed according to standard protocols (41).

PagL mutant plasmid constructs. S. enterica serovar Typhimurium PagL mu-tants were generated by PCR-based overlap extension with Pfu Turbo DNApolymerase (Stratagene). The sequences of the PCR primers are available uponrequest. The expression construct pWKS30-pagL-His6 (27) was used as a tem-plate, and every mutant pagL gene bears a His6 epitope at the C terminus and the79-bp upstream region of pagL. The mutants were cloned into EcoRI/BamHIsites of the low-copy vector pWKS30 (46) or pBluescript II KS(�). The name ofeach mutant construct includes the wild-type residue (single-letter amino aciddesignation) followed by the codon number and mutant residue (typically ala-nine). The amplified insert in the plasmid constructs was verified by DNAsequencing.

Preparation of lipid A. The lipid A used for mass spectrometry was purified asdescribed previously (47). In brief, cells collected from 25 ml of culture wereresuspended in 500 �l of Tri-reagent (Molecular Research Center, Inc., Cincin-nati, OH). After incubation for 15 min at room temperature, 100 �l of chloro-form was added. After 30 min, the mixture was centrifuged, and the aqueousphase was recovered. LPS was extracted three times by the addition of 500 �l ofwater to the organic phase, and the aqueous phase containing LPS was dried witha vacuum concentrator. Five hundred microliters of 10 mM sodium acetatebuffer (pH 4.5) containing 1% sodium dodecyl sulfate (SDS) was add to the driedLPS, and then the LPS was hydrolyzed to remove sugar chains from lipid A byincubation at 95°C for 1 h (39) followed by drying. The dried lipid A was washedtwice with 0.02 N HCl in 99.5% ethanol and three times with 99.5% ethanol. Thewashed lipid A was dried with a vacuum concentrator and then used for massspectrometric analysis.

Alternatively, the lipid A used for the detection of aminoarabinose-modifiedlipid A species was prepared as described previously (7, 25). In brief, LPSpurified from 25 ml of cell culture using an LPS extraction kit (iNtRON Bio-technologies, Inc., Seongnam-Si, Korea) was hydrolyzed for 3 h at 95°C in 150 �lof 100 mM sodium acetate buffer (pH 4.5). Then, 600 �l of a chloroform-methanol mixture (1:2 [vol/vol]), 200 �l of chloroform, and 100 �l of phosphate-buffered saline were added in succession, and the lipid A fraction (chloroformphase) was dried under a stream of nitrogen gas.

Mass spectrometry. Dried lipid A was dissolved in 20 mg/ml of 5-chloro-2-mercaptobenzothiazole matrices in chloroform-methanol (1:1 [vol/vol]). Alter-natively, it was dissolved in chloroform-methanol (1:2 [vol/vol]) for the detectionof aminoarabinose-modified lipid A species and then mixed with 77 mg/ml of2,5-dihydroxybenzoic acid matrices in methanol at a ratio of 1:1. The mixtureswere allowed to dry at room temperature on the sample plate prior to analysis.Spectra were obtained in the negative-reflection mode using a matrix-assistedlaser desorption ionization–time of flight (MALDI-TOF) Voyager-DE STRmass spectrometer (Applied Biosystems Japan, Tokyo, Japan). Each spectrumwas the average of 200 shots. Structural interpretations of lipid A species de-tected by mass spectrometry in this study are summarized in Table 1.

Membrane preparation. All steps were carried out at 4°C or on ice. Cellscollected from 20 ml of bacterial culture were suspended in 300 �l of phosphate-buffered saline and then sonically disrupted three times for 10 s each at 1-minintervals at setting 1 with a Branson sonifier model S-150D. The crude lysate wascleared by centrifugation at 1,000 � g for 5 min. Membranes were precipitatedby centrifugation at 100,000 � g for 30 min and were resuspended in 100 �l ofphosphate-buffered saline. Protein concentrations were determined with thebicinchoninic acid protein assay reagent (Pierce, Rockford, IL) using bovineserum albumin as a standard.

SDS-polyacrylamide gel electrophoresis and Western blotting. Proteins werefractionated by SDS–12.5% polyacrylamide gel electrophoresis under reducingconditions (28). Proteins separated on the gel were stained with Coomassie blue.For the Western blot analysis, proteins separated on the gel were electroblottedonto a nitrocellulose membrane in 25 mM Tris–192 mM glycine–0.02% SDS–20% methanol at 22 V/cm for 60 min. Then the blot was incubated with anti-tetra-His antibodies (Qiagen, Valencia, CA) and subsequently incubated withanti-mouse immunoglobulin G linked to horseradish peroxidase (GE HealthcareBio-Sciences, Piscataway, NJ). Cross-reactive proteins were detected with ECLenhanced chemiluminescence Western blotting detection reagents (GE Health-care Bio-Sciences).

Analysis of LPS by Tricine-SDS-polyacrylamide gel electrophoresis. LPS fromE. coli BL21(DE3) strains was prepared as described previously (13, 22, 43) withslight modifications. In brief, bacterial cells from 1.0 ml of culture, diluted to anoptical density at 600 nm of 1.0, were collected, and suspended in 100 �l ofsample buffer (100 mM Tris-HCl [pH 6.8], 20% glycerol [vol/vol], 4% [wt/vol]SDS, 0.0125% [wt/vol] bromophenol blue, 5% [vol/vol] 2-mercaptoethanol). Thesamples were boiled for 10 min prior to digestion with proteinase K at a finalconcentration of 1 mg/ml for 16 h at 55°C, followed by boiling for 5 min toinactivate proteinase K.

The LPS samples were subjected to Tricine-SDS-polyacrylamide gel electro-phoresis (30). The separating gel was prepared at a final concentration of 18%acrylamide, 1 M Tris-HCl (pH 8.45), 5% glycerol, and 0.05% SDS. The stackinggel was prepared at a final concentration of 4% acrylamide, 1.67 M Tris-HCl (pH8.45), and 0.083% SDS. The samples were loaded under electrophoresis buffer(0.1 M Tris-HCl [pH 8.3], 0.1 M Tricine, 0.1% SDS) and allowed to run at 30 Vfor 50 min and then at 105 V for 215 min. The gels were fixed overnight in 11:8:1(vol/vol/vol) water-ethanol-acetic acid and subsequently stained with a silverstaining kit from Daiichi Pure Chemicals, Tokyo, Japan, according to the man-ufacturer’s instructions.

TABLE 1. Structural interpretations of lipid A species detected bymass spectrometry in this study

m/z Lipid A modification(s)a

1,572 .......................................................Deacylation1,588 .......................................................Deacylation and hydroxylation1,711 .......................................................Deacylation, hydroxylation, and

phosphoethanolamineattachment

1,798 .......................................................Unmodified1,810 .......................................................Deacylation and palmitoylation1,814 .......................................................Hydroxylation1,826 .......................................................Deacylation, palmitoylation,

and hydroxylation1,906 .......................................................Deacylation, palmitoylation and

hydroxylation(pyrophosphate lipid A)b

1,937 .......................................................Hydroxylation andphosphoethanolamineattachment

1,945 .......................................................Aminoarabinose attachmentand hydroxylation

1,957 .......................................................Aminoarabinose attachment,deacylation, palmitoylation,and hydroxylation

2,036 .......................................................Palmitoylation2,052 .......................................................Palmitoylation and

hydroxylation2,132 .......................................................Hydroxylation and

palmitoylation(pyrophosphate lipid A)b

2,175 .......................................................Palmitoylation, hydroxylation,and phosphoethanolamineattachment

a Interpretations were based on previous works (19, 25, 27, 45, 48).b Pyrophosphate lipid A has a pyrophosphate residue instead of a monophos-

phate residue at position 1 of the disaccharide glucosamine of lipid A. Regula-tion of pyrophosphate lipid A synthesis is not well understood (48).

VOL. 190, 2008 RELEASE OF PagL FROM LATENCY BY POINT MUTATIONS 5599


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

RESULTS

Alanine-scanning mutagenesis of extracellular loops of S.enterica PagL. The outer membrane protein PagL was pre-dicted to consist of an eight-stranded �-barrel with four loopsextending into the external environment (13, 40). The PagL ofS. enterica serovar Typhimurium is unique in that it is latent inaminoarabinose-containing outer membranes (27). We specu-lated that the extracellular loops (L1 � L4) of this PagL (Fig.2) sense aminoarabinose-containing outer membranes. Previ-ously, Kawasaki et al. demonstrated that the introduction of alow-copy-vector-based expression construct containing a re-combinant PagL into a S. enterica pmrA pagL mutant, which isdeficient in the aminoarabinose-based modification of lipid A,induced lipid A deacylation, but introduction into an S. entericapmrA� pagL strain did not (27). These results prompted us toscreen for PagL mutants that are no longer latent in S. entericapmrA� strains.

We generated low-copy expression constructs containing mu-tant PagL, in which an amino acid residue located at four loops(L1 � L4) extending into the external environment (Fig. 2) wasreplaced with alanine (Table 2). The expression constructs wereintroduced into an S. enterica pmrA� pagL strain, and the struc-ture of lipid A prepared from the resultant transformants culti-vated in magnesium-limited growth medium, which activates thePhoP-PhoQ two-component regulatory system, was analyzed byMALDI-TOF mass spectrometry. Introduction of expressionconstructs containing most PagL mutants as well as wild-type

PagL induced the production of a negligible or undetectableamount of 3-O-deacylated lipid A species (Fig. 3A and Table 2).In contrast, the introduction of several expression constructs con-taining PagL mutants, such as PagL(R43A), PagL(R44A),PagL(C85A), and PagL(R135A), induced apparent lipid A3-O deacylation (Fig. 3B to E and Table 2). Since aminoarabi-nose-modified lipid A species were not observed well understandard detection conditions for MALDI-TOF mass spec-trometry, as described previously (25), the existence of amino-arabinose-modified lipid A species in the transformants wasconfirmed by using 2,5-dihydroxybenzoic acid as a matrix (insetof Fig. 3). The expression levels of recombinant PagL proteinsin the transformants were confirmed to be similar by Westernblot analysis of the membrane preparations (Fig. 4). Theseresults, taken together, suggest that PagL(R43A), PagL(R44A),PagL(C85A), and PagL(R135A) lost the ability to be latent invivo in the presence of aminoarabinose-modified lipid A species.In addition, the introduction of the PagL(S41A), PagL(I42A),PagL(D83A), PagL(D133A), PagL(V136A), PagL(N137A),and PagL(K172A) mutants into the S. enterica pmrA� pagLstrain induced moderate levels of lipid A 3-O deacylation,suggesting that these mutants also lost the ability to be latentin vivo in the presence of aminoarabinose-modified lipid Aspecies (Table 2).

Arg-43, Arg-44, and Arg-135 are essential for the latency ofS. enterica pagL. Aminoarabinose-modification of lipid A de-creases the net anionic charge at this position and the electrostatic

FIG. 2. Topology model for S. enterica PagL. A model for the topology of S. enterica serovar Typhimurium PagL was constructed based on thesequence similarity to P. aeruginosa PagL (13). The proposed model consists of an eight-stranded �-barrel with four loops (L1 to L4) extendinginto the external environment. Residues in the postulated �-strands are shown in squares. Numbers refer to the position of residues in theprecursor sequence. Asn-21 was identified as the N-terminal amino acid residue of S. enterica PagL after cleavage of the signal peptide (13).



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

repulsion between neighboring LPS molecules (33). It is plausiblethat the positive charges of the Arg-43, Arg-44, and Arg-135residues of S. enterica PagL are involved in the direct interactionwith aminoarabinose-modified lipid A species. In order to exam-

FIG. 3. Introduction of PagL(R43A), PagL(R44A), PagL(C85A),and PagL(R135A) mutants, but not wild-type PagL, induced LPSdeacylation in an S. enterica pmrA� strain. Lipid A prepared from S.enterica serovar Typhimurium pmrA� pagL strain CS283 transformedwith pWKS30 containing wild-type PagL (A), PagL(R43A) (B),PagL(R44A) (C), PagL(C85A) (D), or PagL(R135A) (E) was analyzedby MALDI-TOF mass spectrometry. Insets in panels show results ofMALDI-TOF mass spectrometry of lipid A using 2, 5-dihydroxyben-zoic acid matrices. The m/z values of lipid A species are shown, andthose that represent deacylated lipid A species are denoted by aster-isks. The structural interpretations of lipid A species are summarizedin Table 1. The results are representative of at least two independentexperiments.

TABLE 2. MALDI-TOF mass spectrometry of lipid A prepared fromS. enterica Typhimurium strain CS283 transformed with low-copyexpression vector pWKS30 containing wild-type or mutant PagL

PagL form or mutation Lipid Adeacylation levela

Wild type .......................................................................................................1Mutants

Replacement of amino acidresidue with alanine

Loop 1S41A ...................................................................................................2I42A....................................................................................................2R43A ..................................................................................................4R44A ..................................................................................................3

Loop 2G76A..................................................................................................1F77A...................................................................................................1K78A ..................................................................................................1K80A ..................................................................................................1G81A..................................................................................................1S82A ...................................................................................................1D83A..................................................................................................2D84A..................................................................................................1C85A ..................................................................................................3S86A ...................................................................................................1K87A ..................................................................................................1

Loop 3I126A..................................................................................................1K127A ................................................................................................1S128A .................................................................................................1K129A ................................................................................................1S130A .................................................................................................1R131A ................................................................................................1D132A................................................................................................1D133A................................................................................................2M134A ...............................................................................................1R135A ................................................................................................4V136A ................................................................................................2N137A ................................................................................................2S138A .................................................................................................1F140A.................................................................................................1T141A.................................................................................................1F142A.................................................................................................1

Loop 4N166A ................................................................................................1G167A................................................................................................1S168A .................................................................................................1L169A.................................................................................................1T170A.................................................................................................1D171A................................................................................................1K172A ................................................................................................2N173A ................................................................................................1S174A .................................................................................................1G175A................................................................................................1H176A................................................................................................1N177A ................................................................................................1

Replacement of amino acid residuewith other than alanine

R43K ......................................................................................................3R43H......................................................................................................3R43Q......................................................................................................4R44K ......................................................................................................2C85M......................................................................................................3C85V ......................................................................................................3C85S .......................................................................................................3D133E ....................................................................................................1R135K ....................................................................................................3R135H....................................................................................................3R135Q....................................................................................................3

a Lipid A deacylation levels were determined by MALDI-TOF mass spectrometryand are defined as follows: 1, lipid A deacylation was not observed or a negligiblepeak was detected (Fig. 3A); 2, small but apparent peaks that correspond todeacylated lipid A species are observed (Fig. 5B); 3, approximately half of thepeaks corresponded to deacylated lipid A (Fig. 3C and D and Fig. 5A and C); 4,almost all peaks corresponded to deacylated lipid A species (Fig. 3B and E).



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

ine whether these positive charges are essential for the latency orarginine residues at the positions are essential, we generatedPagL(R43K), PagL(R44K), and PagL(R135K) mutants in whicha cationic arginine residue was replaced with a cationic lysineresidue. The PagL mutants were introduced into the S. entericapmrA� pagL strain, and the structures of lipid A species in thetransformants were analyzed by MALDI-TOF mass spectrome-try. Apparent 3-O-deacylated lipid A species were observed inlipid A prepared from the strains transformed with the PagL(R43K), PagL(R44K), and PagL(R135K) mutants (Fig. 5), sug-gesting that replacement of arginine with lysine at position 43, 44,or 135 resulted in PagL losing the ability to be latent. In addition,apparent 3-O deacylation was also induced by introduction ofPagL(R43H), PagL(R43Q), PagL(R135H), or PagL(R135Q), in-dicating that these mutants also lost the ability to be latent (Table2). Furthermore, PagL(R43K), PagL(R44K), and PagL(R135K)were introduced into the S. enterica pmrA pagL double-mutantstrain, and the lipid A prepared from each transformant wasanalyzed. The introduction of the expression construct containingPagL(R43K), PagL(R44K), or PagL(R135K) induced levels ofdeacylation similar to those in the S. enterica pmrA pagL straintransformed with the expression construct containing wild-typePagL, suggesting that the replacement of arginine with lysine atposition 43, 44, or 135 did not affect the lipid A 3-O-deacylaseactivity (Fig. 6). The levels of recombinant PagL in the strainstransformed with the expression constructs containing wild-typePagL, PagL(R43K), PagL(R44K), and PagL(R135K) were con-firmed to be similar by Western blotting (data not shown). Pre-viously, the lipid A 3-O-deacylase activity of S. enterica PagL wasexamined by heterologous expression in E. coli (13). Therefore, E.coli BL21(DE3) was transformed with the expression constructcontaining wild-type PagL, PagL(R43A), PagL(R44A), or

PagL(R135A), and LPS prepared from the resultant transfor-mants was analyzed by Tricine-SDS-polyacrylamide gel electro-phoresis. Introduction of the PagL(R43A), PagL(R44A), orPagL(R135A) mutant into E. coli BL21(DE3) induced levels ofLPS modification similar to that in the E. coli strain transformedwith wild-type PagL (Fig. 7). These results, taken together, indi-cate that Arg-43, Arg-44, and Arg-135of S. enterica PagL wereessential for the latency, and mutations at these positions did notaffect the lipid A 3-O-deacylase activity. The importance of thearginine residues could not simply be attributed to their positivecharge.

In addition, to evaluate the importance of Cys-85 for latency,PagL(C85M), PagL(C85V), and PagL(C85S) mutants weregenerated. Apparent 3-O-deacylated lipid A species were ob-served in S. enterica transformed with the expression constructcontaining the PagL(C85M), PagL(C85V), or PagL(C85S) mu-tant (Table 2). In addition, introduction of PagL(C85A) into E.coli BL21(DE3) induced the modification of LPS to levelssimilar to that in E. coli transformed with wild-type PagL (Fig.7). These results, taken together, indicate that Cys-85 is in-volved in latency.

Asn-173 and Asn-177 of PagL were essential for lipid A3-O-deacylase activity. The low-copy expression constructscontaining mutants, in which an amino acid residue located atthe extracellular loops (L1 � L4) was replaced with alanine,were introduced into the S. enterica pmrA pagL strain, in whichlipid A is not modified by aminoarabinose. Lipid A was pre-

FIG. 4. Expression levels of recombinant PagL proteins were similaramong S. enterica pmrA� strains transformed with expression constructscontaining wild-type PagL, PagL(R43A), PagL(R44A), PagL(C85A), orPagL(R135A). Ten-microgram samples of membrane proteins preparedfrom S. enterica serovar Typhimurium pmrA� pagL strain CS283 trans-formed with the pWKS30 vector (vector) or pWKS30 containing wild-type PagL, PagL(R43A), PagL(R44A), PagL(C85A), or PagL(R135A)were subjected to SDS–12.5% polyacrylamide gel electrophoresis andanalyzed by staining with Coomassie blue (A) or by Western-blottingusing anti-tetra-His antibody (B).

FIG. 5. Introduction of PagL(R43K), PagL(R44K), and PagL(R135K) induced LPS deacylation in an S. enterica pmrA� strain. LipidA prepared from S. enterica serovar Typhimurium pmrA� pagL strainCS283 transformed with pWKS30 containing PagL(R43K) (A),PagL(R44K) (B), or PagL(R135K) (C) was analyzed by MALDI-TOFmass spectrometry. The m/z values of lipid A species are shown, andthose that represent deacylated lipid A species are denoted by aster-isks. The structural interpretations of lipid A species are summarizedin Table 1. The results are representative of at least two independentexperiments.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

pared from the transformants, and its structure was analyzedby MALDI-TOF mass spectrometry. The analysis revealedthat the PagL(N173A) and PagL(N177A) mutants did not in-duce the deacylation of lipid A (Fig. 8). Expression levels ofthe mutant PagL proteins were similar to that of wild-typerecombinant PagL protein (data not shown), suggesting thatthe PagL mutants lost lipid A 3-O-deacylase activity. In addi-tion, introduction of expression constructs containing PagL(N173A) or PagL(N177A) into E. coli BL21(DE3) did notinduce modification of LPS (Fig. 7). These results, taken to-gether, indicate that Asn-173 and Asn-177 were essential forthe lipid A 3-O-deacylase activity of PagL. Asn-173 and Asn-177 of S. enterica PagL correspond to Asn-159 (136 from pre-dicted N terminus) and Glu-163 (140 from predicted N termi-nus) of P. aeruginosa PagL, which were previously demonstratedto be important for the lipid A 3-O-deacylase activity of P. aerugi-nosa PagL (40). In addition, previous reports demonstrated that

P. aeruginosa PagL Asn-152 (129 from predicted N terminus),which corresponds to Asn-166 of S. enterica PagL, was impor-tant for PagL activity (40). However, the introduction of ex-pression constructs containing S. enterica PagL(N166A) intothe S. enterica pmrA pagL or E. coli BL21(DE3) strain inducedthe modification of LPS (Fig. 6 and 7), suggesting that Asn-166is not crucial for the lipid A 3-O-deacylase activity of S. entericaPagL. Introduction of other low-copy expression constructscontaining mutant PagL, in which an amino acid residue lo-cated in the four extracellular loops (Fig. 2) was replaced withalanine, induced lipid A deacylation in the S. enterica pmrAstrain (data not shown), suggesting other amino acid residueslocated at extracellular loops of PagL not to be essential for the3-O-deacylase activity.

S. enterica strains expressing PagL mutants that lost thelatency showed growth arrest at 43°C. In order to examinethe physiological significance of PagL’s latency, we analyzedthe growth rates of S. enterica strains expressing PagL mutantsthat had lost latency. S. enterica pmrA� pagL cells transformedwith low-copy expression constructs containing PagL(R43A)or PagL(R135A), which lost latency as described above, had agrowth rate at 37°C similar to that of S. enterica transformedwith low-copy expression constructs containing wild-type PagL(Fig. 9A). In contrast, S. enterica pmrA� pagL cells trans-formed with the expression construct containing PagL(R43A)or PagL(R135A) showed apparent growth arrest at 43°C com-pared with that of S. enterica transformed with the expressionconstruct containing wild-type PagL (Fig. 9B). In addition, theS. enterica pmrA� pagL strain transformed with the expressionconstruct containing PagL(R131A), which retains the ability tobe latent (Table 2), did not show growth arrest at 43°C (Fig.9B). These results, taken together, suggest that the amino-arabinose modification-dependent latency of PagL is impor-tant for cell growth at 43°C.

DISCUSSION

We demonstrated that mutations of several amino acid res-idues located at extracellular loops in the PagL of S. enterica

FIG. 6. Introduction of PagL(R43K), PagL(R44K), and PagL(R135K) mutants into an S. enterica pmrA mutant strain induced LPSdeacylation to similar levels as those induced by introduction of wild-typePagL. Lipid A prepared from S. enterica serovar Typhimurium pmrA pagLdouble-mutant strain KCS040 transformed with pWKS30 containing wild-type PagL (A), PagL(R43K) (B), PagL(R44K) (C), or PagL(R135K)(D) was analyzed by MALDI-TOF mass spectrometry. The m/z values oflipid A species are shown, and those that represent deacylated lipid Aspecies are denoted by asterisks. The structural interpretations of lipid Aspecies are summarized in Table 1. The results are representative of atleast two independent experiments.

FIG. 7. Analysis of LPS modification in E. coli transformed with PagLmutant by Tricine-SDS-polyacrylamide gel electrophoresis. LPS preparedfrom E. coli BL21(DE3) transformed with pBluescript II KS(�) (vector)or pBluescript II KS(�) containing wild-type PagL, PagL(R43A), PagL(R44A), PagL(C85A), PagL(R135A), PagL(N166A), PagL(N173A), orPagL(N177A) was analyzed by Tricine-SDS-polyacrylamide gel electro-phoresis. The results are representative of at least two independent ex-periments.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

serovar Typhimurium, including Arg-43, Arg-44, Cys-85, andArg-135, released PagL from the aminoarabinose modifica-tion-dependent latency. These mutations were distinct fromthose that affected the lipid A 3-O-deacylase activity of PagL.Furthermore, S. enterica cells expressing mutant PagL that lostlatency exhibited growth arrest at 43°C. These observationsindicate that the extracellular loops of S. enterica PagL areinvolved in recognition of aminoarabinose-modified outermembranes, and this recognition is important for bacterialgrowth at 43°C.

The modification of lipid A by aminoarabinose changes thecell surface membrane charge, since the primary amine ofaminoarabinose possesses a positive charge (Fig. 1). Thechanges are known to increase bacterial resistance to cationicantimicrobial peptides, including polymyxin B (17, 18, 29, 34).The involvement of cationic amino acid residues, such as Arg-43, Arg-44, and Arg-135, in latency suggests some direct elec-trostatic interaction between the positively charged domain ofPagL’s extracellular loops and cell surface aminoarabinose-

modified lipid A, although the replacement of Arg-43, Arg-44,and Arg-135 of PagL with cationic lysine residues did notsustain the latency. PagL was latent in S. enterica (27, 45) butnot in Pseudomonas aeruginosa (8, 10). Consistent with ourobservations, the Arg-43, Arg-44, Cys-85, and Arg-135 residuesof S. enterica PagL, which were essential for latency, were notconserved in P. aeruginosa PagL (13, 45). The crystal structureof P. aeruginosa PagL reveals that its active site faces the outersurface of the outer membrane (40). The potential of PagL toform a dimer at the interface within the active sites suggests apossible mechanism to inhibit the activity of S. enterica PagL inthe outer membrane (40). Modification of the outer membraneby aminoarabinose as well as amino acid residues located atextracellular loops of PagL, such as Arg-43, Arg-44, Cys-85,and Arg-135, might be involved in the structural changes ofPagL. The structural changes, which make PagL silent in theouter membrane, remain to be elucidated.

In addition to the amino acid residues involved in the sens-ing of aminoarabinose-containing membranes, we showed thatAsn-173 and Asn-177 located at extracellular loops of S. en-terica PagL were essential for the lipid A 3-O-deacylase activ-ity. These results were consistent with previous findings on P.aeruginosa PagL (13). On the other hand, the replacement ofAsn-166 of S. enterica PagL with alanine did not abolish thedeacylase activity, a result not consistent with the report thatthe replacement of Asn-152 (129 from predicted N terminus)of P. aeruginosa PagL, which corresponds to Asn-166 of S.

FIG. 8. Asn-173 and Asn-177 are essential for lipid A 3-O-deacyl-ase activity of PagL in an S. enterica pmrA mutant strain. Lipid Aprepared from S. enterica serovar Typhimurium pmrA pagL double-mutant strain KCS040 transformed with the pWKS30 vector (A) orpWKS30 containing PagL(N166A) (B), PagL(N173A) (C), orPagL(N177A) (D) was analyzed by MALDI-TOF mass spectrometry.The m/z values of lipid A species are shown, and those that representdeacylated lipid A species are denoted by asterisks. The structuralinterpretations of lipid A species are summarized in Table 1. Theresults are representative of at least two independent experiments.

FIG. 9. Introduction of PagL(R43A) and PagL(R135A) mutantsinto S. enterica induced growth arrest at 43°C. S. enterica serovarTyphimurium pmrA� pagL strain CS283 transformed with pWKS30(vector) or pWKS30 containing wild-type PagL, PagL(R43A),PagL(R131A), or PagL(R135A) was grown overnight at 37°C. Thenthe cells were diluted to an optical density at 600 nm of 0.05 and grownat 37°C (A) or 43°C (B). Cell growth was measured by monitoringoptical density at 600 nm. The results are representative of two (A) orthree (B) independent experiments.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

enterica PagL, with alanine abolished the lipid A 3-O-deacylaseactivity (13). The discrepancy suggests some structural differ-ence in the active sites between S. enterica PagL and P. aerugi-nosa PagL.

In this study, we observed that S. enterica expressing thePagL-R43A or PagL-R135A mutant showed growth arrest at43°C. These results suggest PagL’s latency to help S. enterica togrow under specific conditions, including in the tissues of ahost who has a fever, and are the first observations to suggestthe physiological importance of PagL’s latency. Several otherouter membrane enzymes involved in the modification of lipidA, such as S. enterica LpxR and E. coli PagP, also displaylatency (5); LpxR-dependent lipid A deacylation in S. enterica(38) and PagP-dependent lipid A palmitoylation in E. coli (23,42) were not usually observed under normal culture conditions.These observations suggest that the latency of outer membraneenzymes is generally conserved for regulation of lipid A mod-ifications in gram-negative bacteria. Although little is knownabout the physiological functions of the repression of outermembrane enzymes involved in the modification of lipid A, ourresults regarding PagL suggest that the latency of these en-zymes is involved in bacterial pathogenesis.

ACKNOWLEDGMENT

This work was supported in part by a Grant-in-Aid for ScientificResearch from the Japan Society for the Promotion of Science.

REFERENCES

1. Bader, M. W., W. W. Navarre, W. Shiau, H. Nikaido, J. G. Frye, M. McClelland,F. C. Fang, and S. I. Miller. 2003. Regulation of Salmonella typhimuriumvirulence gene expression by cationic antimicrobial peptides. Mol. Microbiol.50:219–230.

2. Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E.Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobialpeptides by a bacterial sensor kinase. Cell 122:461–472.

3. Bearson, B. L., L. Wilson, and J. W. Foster. 1998. A low pH-inducible,PhoPQ-dependent acid tolerance response protects Salmonella typhimuriumagainst inorganic acid stress. J. Bacteriol. 180:2409–2417.

4. Belden, W. J., and S. I. Miller. 1994. Further characterization of the PhoPregulon: identification of new PhoP-activated virulence loci. Infect. Immun.62:5095–5101.

5. Bishop, R. E. 2007. Structural biology of membrane-intrinsic beta-barrelenzymes: sentinels of the bacterial outer membrane. Biochim. Biophys. Actadoi:10.1016/j.bbamem.2007.07.021.

6. Bishop, R. E., H. S. Gibbons, T. Guina, M. S. Trent, S. I. Miller, and C. R.Raetz. 2000. Transfer of palmitate from phospholipids to lipid A in outermembranes of gram-negative bacteria. EMBO J. 19:5071–5080.

7. Brett, P. J., M. N. Burtnick, D. S. Snyder, J. G. Shannon, P. Azadi, and F. C.Gherardini. 2007. Burkholderia mallei expresses a unique lipopolysaccha-ride mixture that is a potent activator of human Toll-like receptor 4 com-plexes. Mol. Microbiol. 63:379–390.

8. Ernst, R. K., K. N. Adams, S. M. Moskowitz, G. M. Kraig, K. Kawasaki,C. M. Stead, M. S. Trent, and S. I. Miller. 2006. The Pseudomonas aerugi-nosa lipid A deacylase: selection for expression and loss within the cysticfibrosis airway. J. Bacteriol. 188:191–201.

9. Ernst, R. K., T. Guina, and S. I. Miller. 2001. Salmonella typhimurium outermembrane remodeling: role in resistance to host innate immunity. MicrobesInfect. 3:1327–1334.

10. Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I.Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airwayPseudomonas aeruginosa. Science 286:1561–1565.

11. Fields, P. I., E. A. Groisman, and F. Heffron. 1989. A Salmonella locus thatcontrols resistance to microbicidal proteins from phagocytic cells. Science243:1059–1062.

12. Garcia Vescovi, E., F. C. Soncini, and E. A. Groisman. 1996. Mg2� as anextracellular signal: environmental regulation of Salmonella virulence. Cell84:165–174.

13. Geurtsen, J., L. Steeghs, J. T. Hove, P. van der Ley, and J. Tommassen. 2005.Dissemination of lipid A deacylases (pagL) among gram-negative bacteria:identification of active-site histidine and serine residues. J. Biol. Chem.280:8248–8259.

14. Gibbons, H. S., S. R. Kalb, R. J. Cotter, and C. R. Raetz. 2005. Role of Mg2�

and pH in the modification of Salmonella lipid A after endocytosis bymacrophage tumour cells. Mol. Microbiol. 55:425–440.

15. Gibbons, H. S., S. Lin, R. J. Cotter, and C. R. Raetz. 2000. Oxygen require-ment for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonellatyphimurium lipid A. Function of LpxO, A new Fe2�/alpha-ketoglutarate-dependent dioxygenase homologue. J. Biol. Chem. 275:32940–32949.

16. Groisman, E. A., E. Chiao, C. J. Lipps, and F. Heffron. 1989. Salmonellatyphimurium phoP virulence gene is a transcriptional regulator. Proc. Natl.Acad. Sci. USA 86:7077–7081.

17. Gunn, J. S., K. B. Lim, J. Krueger, K. Kim, L. Guo, M. Hackett, and S. I.Miller. 1998. PmrA-PmrB-regulated genes necessary for 4-aminoarabinoselipid A modification and polymyxin resistance. Mol. Microbiol. 27:1171–1182.

18. Gunn, J. S., and S. I. Miller. 1996. PhoP-PhoQ activates transcription ofpmrAB, encoding a two-component regulatory system involved in Salmonellatyphimurium antimicrobial peptide resistance. J. Bacteriol. 178:6857–6864.

19. Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller.2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus nec-essary for lipopolysaccharide modification, antimicrobial peptide resistance,and oral virulence of Salmonella enterica serovar Typhimurium. Infect. Im-mun. 68:6139–6146.

20. Guo, L., K. B. Lim, J. S. Gunn, B. Bainbridge, R. P. Darveau, M. Hackett,and S. I. Miller. 1997. Regulation of lipid A modifications by Salmonellatyphimurium virulence genes phoP-phoQ. Science 276:250–253.

21. Guo, L., K. B. Lim, C. M. Poduje, M. Daniel, J. S. Gunn, M. Hackett, andS. I. Miller. 1998. Lipid A acylation and bacterial resistance against verte-brate antimicrobial peptides. Cell 95:189–198.

22. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneityamong Salmonella lipopolysaccharide chemotypes in silver-stained poly-acrylamide gels. J. Bacteriol. 154:269–277.

23. Jia, W., A. El Zoeiby, T. N. Petruzziello, B. Jayabalasingham, S.Seyedirashti, and R. E. Bishop. 2004. Lipid trafficking controls endotoxinacylation in outer membranes of Escherichia coli. J. Biol. Chem. 279:44966–44975.

24. Kato, A., and E. A. Groisman. 2004. Connecting two-component regulatorysystems by a protein that protects a response regulator from dephosphory-lation by its cognate sensor. Genes Dev. 18:2302–2313.

25. Kawasaki, K., K. China, and M. Nishijima. 2007. Release of the lipopolysac-charide deacylase PagL from latency compensates for a lack of lipopolysaccha-ride aminoarabinose modification-dependent resistance to the antimicrobialpeptide polymyxin B in Salmonella enterica. J. Bacteriol. 189:4911–4919.

26. Kawasaki, K., R. K. Ernst, and S. I. Miller. 2004. 3-O-deacylation of lipid Aby PagL, a PhoP/PhoQ-regulated deacylase of Salmonella typhimurium,modulates signaling through Toll-like receptor 4. J. Biol. Chem. 279:20044–20048.

27. Kawasaki, K., R. K. Ernst, and S. I. Miller. 2005. Inhibition of Salmonellaenterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabi-nose membrane modification. J. Bacteriol. 187:2448–2457.

28. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

29. Lee, H., F. F. Hsu, J. Turk, and E. A. Groisman. 2004. The PmrA-regulatedpmrC gene mediates phosphoethanolamine modification of lipid A and poly-myxin resistance in Salmonella enterica. J. Bacteriol. 186:4124–4133.

30. Lesse, A. J., A. A. Campagnari, W. E. Bittner, and M. A. Apicella. 1990.Increased resolution of lipopolysaccharides and lipooligosaccharides utiliz-ing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Im-munol. Methods 126:109–117.

31. Miller, S. I., A. M. Kukral, and J. J. Mekalanos. 1989. A two-componentregulatory system (phoP phoQ) controls Salmonella typhimurium virulence.Proc. Natl. Acad. Sci. USA 86:5054–5058.

32. Miyake, K. 2004. Innate recognition of lipopolysaccharide by Toll-like re-ceptor 4-MD-2. Trends Microbiol. 12:186–192.

33. Nikaido, H. 2003. Molecular basis of bacterial outer membrane permeabilityrevisited. Microbiol. Mol. Biol. Rev. 67:593–656.

34. Nummila, K., I. Kilpelainen, U. Zahringer, M. Vaara, and I. M. Helander.1995. Lipopolysaccharides of polymyxin B-resistant mutants of Escherichiacoli are extensively substituted by 2-aminoethyl pyrophosphate and containaminoarabinose in lipid A. Mol. Microbiol. 16:271–278.

35. Prost, L. R., M. E. Daley, V. Le Sage, M. W. Bader, H. Le Moual, R. E. Klevit,and S. I. Miller. 2007. Activation of the bacterial sensor kinase PhoQ byacidic pH. Mol. Cell 26:165–174.

36. Raetz, C. R., C. M. Reynolds, M. S. Trent, and R. E. Bishop. 2007. Lipid Amodification systems in gram-negative bacteria. Annu. Rev. Biochem. 76:295–329.

37. Raetz, C. R., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu.Rev. Biochem. 71:635–700.

38. Reynolds, C. M., A. A. Ribeiro, S. C. McGrath, R. J. Cotter, C. R. Raetz, andM. S. Trent. 2006. An outer membrane enzyme encoded by Salmonellatyphimurium lpxR that removes the 3�-acyloxyacyl moiety of lipid A. J. Biol.Chem. 281:21974–21987.

39. Rosner, M. R., J. Tang, I. Barzilay, and H. G. Khorana. 1979. Structure ofthe lipopolysaccharide from an Escherichia coli heptose-less mutant. I.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Chemical degradations and identification of products. J. Biol. Chem. 254:5906–5917.

40. Rutten, L., J. Geurtsen, W. Lambert, J. J. Smolenaers, A. M. Bonvin, A. deHaan, P. van der Ley, M. R. Egmond, P. Gros, and J. Tommassen. 2006.Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagLfrom Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 103:7071–7076.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, NY.

42. Smith, A. E., S. H. Kim, F. Liu, W. Jia, E. Vinogradov, C. L. Gyles, and R. E.Bishop. 2008. PagP activation in the outer membrane triggers R3 coreoligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7.J. Biol. Chem. 283:4332–4343.

43. Steiner, K., R. Novotny, K. Patel, E. Vinogradov, C. Whitfield, M. A. Val-vano, P. Messner, and C. Schaffer. 2007. Functional characterization of theinitiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillusstearothermophilus NRS 2004/3a. J. Bacteriol. 189:2590–2598.

44. Tanamoto, K., and S. Azumi. 2000. Salmonella-type heptaacylated lipid A is

inactive and acts as an antagonist of lipopolysaccharide action on human linecells. J. Immunol. 164:3149–3156.

45. Trent, M. S., W. Pabich, C. R. Raetz, and S. I. Miller. 2001. A PhoP/PhoQ-induced lipase (PagL) that catalyzes 3-O-deacylation of lipid Aprecursors in membranes of Salmonella typhimurium. J. Biol. Chem.276:9083–9092.

46. Wang, R. F., and S. R. Kushner. 1991. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichiacoli. Gene 100:195–199.

47. Yi, E. C., and M. Hackett. 2000. Rapid isolation method for lipopolysaccha-ride and lipid A from gram-negative bacteria. Analyst 125:651–656.

48. Zhou, Z., S. Lin, R. J. Cotter, and C. R. Raetz. 1999. Lipid A modificationscharacteristic of Salmonella typhimurium are induced by NH4VO3 in Esch-erichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoeth-anolamine and palmitate. J. Biol. Chem. 274:18503–18514.

49. Zhou, Z., A. A. Ribeiro, S. Lin, R. J. Cotter, S. I. Miller, and C. R. Raetz. 2001.Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorpora-tion. J. Biol. Chem. 276:43111–43121.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Documents

Extracellular Loops of Lipid A 3-O-Deacylase PagL Are ...in which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipid A 3-O deacylation