functional analysis of the large periplasmic loop of the escherichia coli

Functional analysis of the large periplasmic loop of theEscherichia coli K-12 WaaL O-antigen ligase

José M. Pérez, Megan A. McGarry,Cristina L. Marolda and Miguel A. Valvano*Infectious Diseases Research Group, Siebens-DrakeResearch Institute, Department of Microbiology andImmunology, University of Western Ontario, London,N6A 5C1, Canada.

Summary

WaaL is a membrane enzyme implicated in ligatingundecaprenyl-diphosphate (Und-PP)-linked O antigento lipid A-core oligosaccharide. We determined theperiplasmic location of a large (EL5) and small (EL4)adjacent loops in the Escherichia coli K-12 WaaL.Structural models of the EL5 from the K-12, R1 andR4 E. coli ligases were generated by moleculardynamics. Despite the poor amino acid sequenceconservation among these proteins, the modelsafforded similar folds consisting of two pairs ofalmost perpendicular a-helices. One a-helix in eachpair contributes a histidine and an arginine facingeach other, which are highly conserved in WaaLhomologues. Mutations in either residue renderedWaaL non-functional, since mutant proteins wereunable to restore O antigen surface expression.Replacements of residues located away from theputative catalytic centre and non-conserved residueswithin the centre itself did not affect ligation. Further-more, replacing a highly conserved arginine in EL4with various amino acids inactivates WaaL function,but functionality reappears when the positive chargeis restored by a replacement with lysine. Theseresults lead us to propose that the conserved aminoacids in the two adjacent periplasmic loops couldinteract with Und-PP, which is the common compo-nent in all WaaL substrates.

Introduction

Gram-negative bacteria have an asymmetric outer mem-brane bilayer rich in phospholipids on the inner leaflet andlipopolysaccharide (LPS) on the outer leaflet (Kamio and

Nikaido, 1976; Nikaido, 2003). LPS is key to the effectivepermeability barrier properties of the outer membrane(reviewed in Nikaido, 2003) and consists of lipid A, coreoligosaccharide (OS) and the O polysaccharide or Oantigen (reviewed in Raetz and Whitfield, 2002; Valvano,2003). Prior to ligation, the initial synthesis of lipid A-coreOS and O antigen polymers occurs independently at thecytoplasmic side of the inner membrane (Raetz and Whit-field, 2002; Valvano, 2003). The lipid A-core OS is trans-located to the periplasmic site of the membrane by aprocess that requires ATP hydrolysis, mediated bythe ABC transporter MsbA (Doerrler et al., 2001; Doerrlerand Raetz, 2002). The O antigen is synthesized as anundecaprenyl-diphosphate (Und-PP)-linked intermediate,and translocated across the membrane by the Wzy-, ABCtransporter- or synthase-dependent pathways (Raetz andWhitfield, 2002). The Wzy-dependent pathway requiresthe membrane proteins Wzx (Und-PP-O antigen flippase),Wzy (O antigen polymerase) and Wzz (regulator ofO antigen polysaccharide chain length distribution)(Raetz and Whitfield, 2002; Valvano, 2003). The ABCtransporter-dependent pathway employs the proteinsWzm and Wzt, which are the permease and ATPhydrolysis components of an ABC-class 2 transporterrespectively (Raetz and Whitfield, 2002). The synthase-dependent pathway occurs in Salmonella entericaserovar Borreze (Keenleyside and Whitfield, 1996) andrequires a single protein of the hyaluronan synthasefamily (Raetz and Whitfield, 2002).

Irrespective of the export and polymerization modes ofthe saccharide molecules, nascent Und-PP-linked O anti-gens are ligated to terminal sugar residues of the lipidA-core OS (McGrath and Osborn, 1991) in a reactionmediated by the membrane protein WaaL. Mutations inthe waaL gene prevent the ligation of O antigen moleculesto lipid A-core OS, resulting in bacteria producing a ‘rough’LPS (lacking O antigen polysaccharide) and accumulatingintracellular Und-PP-linked O antigen molecules (Mulfordand Osborn, 1983; McGrath and Osborn, 1991). Since Oantigens of pathogenic bacteria are usually required forresistance to complement-mediated killing (Pluschke andAchtman, 1984; Joiner, 1988), the elucidation of the ligasereaction could potentially be exploited for the discovery ofnovel LPS biosynthesis inhibitors. However, the mecha-nism of ligation is still unresolved. Although the ligase

Accepted 1 October, 2008. *For correspondence. E-mail [email protected]; Tel. (+1) 519 661 3427; Fax (+1) 519 661 3499.

Molecular Microbiology (2008) 70(6), 1424–1440 � doi:10.1111/j.1365-2958.2008.06490.xFirst published online 29 October 2008

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

catalyses the formation of a glycosidic bond, WaaL pro-teins bear no relationship with classical glycosyltrans-ferases, which modify sugar nucleotide substrates. Theligation reaction shows no specificity for the type of Oantigen or the mechanism of O antigen export (Raetz andWhitfield, 2002). In contrast, a requirement for a specificlipid A-core OS acceptor structure has been established inseveral models (Heinrichs et al., 1998a,b; Abeyrathneet al., 2005; Schild et al., 2005), which has led to thegeneralized notion that a specific WaaL protein recog-nizes a given core OS terminal structure. However, itis unclear how WaaL recognizes the Und-PP-linked Oantigen and, in particular, which part of this moleculeparticipates in the enzymatic reaction.

WaaL proteins show significant divergence in theirprimary amino acid sequence, even for members from thesame species (Raetz et al., 2007). The poor sequenceconservation among O antigen ligases makes compara-tive analyses difficult. It is also difficult to establish rela-tionships in WaaL proteins based on potential core OSacceptor structures. For example, the Escherichia coli R2and S. enterica WaaL proteins share ~80% amino acidsequence similarity and are functionally interchangeable,as both link the O antigen polysaccharide to a terminalglucose in the core OS that has an a-1,2-linkedN-acetylglucosamine (Heinrichs et al., 1998a). In con-trast, E. coli R3 WaaL is ~66% similar to the Salmonellaprotein but links the O antigen polysaccharide to a differ-ent site of attachment in the core OS that resembles asimilar site in the K-12 core OS. However, the R3 WaaLshares very little identity with the E. coli K-12 ligase (Hei-nrichs et al., 1998c). More recent evidence suggests thatthe specificity of the ligation reaction for a particular corelipid A-core OS structure does not solely depend on theWaaL protein, but presumed additional factor or factorshave not been identified (Kaniuk et al., 2004).

Conceivably, WaaL activity requires amino acidsexposed to the periplasmic space where they could inter-act with donor and acceptor molecules. A critical Hisresidue was identified in a periplasmic loop of the Vibriocholerae WaaL (Schild et al., 2005), and a potentiallycommon motif is emerging not only in WaaL proteins butalso in proteins that ligate Und-PP-linked O antigen pre-cursors to pili (Qutyan et al., 2007). Recently, Abeyrathneand Lam (2007) reported that highly purified WaaL fromPseudomonas aeruginosa has ATPase activity and ATPhydrolysis is required for the in vitro ligation reaction. Thisis an intriguing finding since ATP is not present in theperiplasmic space (Pugsley, 1993).

In this work, we established a topological model of theE. coli K-12 WaaL ligase and identified highly conservedresidues in two adjacent periplasmic loops, which arerequired for function. Using molecular dynamics, struc-tural models of one of these loops were constructed for

the E. coli K-12, R1 and R4 WaaL proteins. All modelsrevealed a similar fold consisting of two pairs of almostperpendicular a-helices. Highly conserved histidine andarginine residues, required for WaaL function, are locatedfacing each other in the cross-over region between onea-helix of each pair both. Our results provide a predictivemodel to probe structure–function relationships in WaaLproteins and lead us to propose the existence of a cata-lytic region that could interact with Und-PP, the commonmolecule in the Und-PP-linked O-antigen substrates forthe ligation reaction.

Results

Predicted topology of E. coli K-12 WaaL

Using current algorithms for predicting transmembranehelices and in–out topologies of the intervening loops(Nilsson et al., 2000; Drew et al., 2002), we constructed atopological model for the E. coli K-12 WaaL protein (419amino acids) that consists of 12 predicted transmembranedomains, six periplasmic loops, five cytoplasmic loops,and has the N- and C-termini in the cytoplasm (Fig. 1). Weinvestigated the orientation of the WaaL C-terminus usingthe green fluorescent protein (GFP) as a topology probe,since GFP can only stably fold into a chromophore whenpresent in the cytoplasm (Feilmeier et al., 2000; Drewet al., 2002). E. coli DH5a transformants containingpMM4 (which encodes WaaL C-terminally fused to GFP atamino acid 419; WaaLGFP; Table 1) expressed a polypep-tide with an apparent mass of 70 kDa, in agreement to thepredicted molecular mass of the WaaLGFP fusion protein(Fig. 2A, lane 1), and exhibited green fluorescence local-ized to the periphery of each cell (Fig. 2B). Additionalbands of lower molecular mass reacting with the anti-GFPM2 monoclonal antibody (Fig. 2A, lane 1) were inter-preted as degradation products. In the control experiment,cells containing pBADGFP expressed a ~30 kDa polypep-tide (Fig. 2A, lane 2), similar to the approximate molecularmass of GFPmut3 (27 kDa), and displayed green fluores-cence uniformly distributed throughout the cell body(Fig. 2C). We conclude from this experiment that theWaaL C-terminus resides in the cytosol.

The functionality of WaaLGFP was assessed using thewaaL-deficient strain CLM24(pMF19) (Feldman et al.,2005). Plasmid pMF19 encodes a rhamnosyltransferasethat allows for the completion of the O16 subunit synthe-sis in E. coli K-12 strains of the W3110 lineage (Liu andReeves, 1994; Feldman et al., 1999). LPS samples pre-pared from CLM24(pMF19) containing pMM4 revealedthe classical ladder-like pattern of O16 antigen polysac-charide, with lower bands corresponding to lipid A-coreOS (core) and lipid A-core OS plus one O unit (core + 1),and higher molecular weight bands corresponding to lipid

Functional analysis of the WaaL large periplasmic loop 1425

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 70, 1424–1440

A-core OS ligated to polymeric O antigen (Fig. 3A, lane1). A similar result was obtained with CLM24(pMF19)containing pCM234 (expressing WaaL with a C-terminalFLAG fusion; Fig. 3A, lane 4) or pCM235 (expressingWaaL with a C-terminal FLAG-5xHis fusion; data notshown). The negative control strain containing the pBAD-FLAG cloning vector did not produce LPS O antigen(Fig. 3A, lane 2). Therefore, the WaaL C-terminus residesin the cytosol and can also tolerate protein fusions withouta noticeable effect in WaaL function.

The external loop 5 is required for WaaL function andfaces the periplasm

All WaaL homologues have a predicted large periplasmicloop of variable length (Schild et al., 2005; Abeyrathneand Lam, 2007; J.M. Pérez and M.A. Valvano,unpublished). In WaaLEcK12 this loop (external loop 5, EL5)has 83 amino acids between Asn-259 and Glu-342(Fig. 1). To investigate the functional role of EL5 in LPS Oantigen synthesis, we constructed pMM1. This plasmidencodes a deletion derivative of WaaL (WaaLDEL5) thatlacks EL5 and also 5 amino acids from the TM10 (Asn-

259 to Lys-347, Fig. 1). The last 5 amino acids of TM10were included in the deletion since the precise EL5–TM10boundary was not certain and lysine residues are nottypical in TM helices. CLM24(pMF19, pMM1) did notproduce LPS O antigen (Fig. 3A, lane 3), despite that theprotein was expressed and correctly localized to themembrane (Fig. 3B, lane 2). Therefore, loss of the largeperiplasmic loop does not affect membrane localizationand/or WaaL expression, but an intact loop is required forligation.

To experimentally address the location of EL5, we con-structed a C-terminal GFP fusion after Arg-265 (Fig. 1).Although the WaaL1-R265-GFP derivative was detected byWestern blotting, E. coli DH5a expressing WaaL1-R265-GFP

(pMM3) did not exhibit green fluorescence (data notshown), suggesting that the fused GFP moiety resides inthe periplasm. The periplasmic location of EL5 was alsoinvestigated in the full-length WaaL by a protease protec-tion assay using intact spheroplasts of E. coli cellsexpressing WaaLFLAG (pCM234). Spheroplasts are usefulfor membrane topology studies since the disruption of theouter membrane exposes the periplasmic domains ofplasma membrane proteins, which become accessible to

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Fig. 1. Topological model of the E. coli K-12 WaaL ligase. The model was originally derived according to the TMHHM computer program(Sonnhammer et al., 1998). The residues spanning predicted transmembrane segments are within rectangular boxes. EL5, external loop 5.Residues in black and white background correspond to those that when replaced by alanine result in null or reduced WaaL functionrespectively (except for P303, see Results). Thick bars indicate the end-points of the deleted region in WaaLDEL5 (pMM1).

1426 J. M. Pérez, M. A. McGarry, C. L. Marolda and M. A. Valvano �


proteolysis (Dai et al., 1996; Haardt and Bremer, 1996;Raza et al., 1996; Benoit et al., 1998). We employedtrypsin, which cleaves at lysine and arginine residues.E. coli K-12 WaaL contains seven predicted trypsin cleav-age sites within the EL5, one within EL3, EL4 and EL6,and two in EL2, while the remaining 20 sites are in pre-dicted cytoplasmic regions of the protein or in transmem-brane domains (data not shown). The FLAG epitopeallows detection of cleavage products containing an intactC-terminus. Thus, trypsin digestion of a periplasmic-exposed EL5 in intact spheroplasts should yieldC-terminal products ranging from 20 to 22 kDa that can be

detected with anti-FLAG antibodies. Spheroplasts of cellsexpressing WaaLFLAG were isolated and incubated withtrypsin for 2 or 4 h. Polypeptides of apparent masses of19, 25 and 50 kDa were observed at 4 h digestion, whichagree with the predicted molecular weights of cleavageproducts at EL5 and EL4, and the full-length undigestedWaaL respectively (Fig. 4A, lane 2). Cleavage productscontaining the FLAG epitope were absent without trypsinor after 2 h digestion (Fig. 4A, lanes 3 and 4). As a control,spheroplasts were lysed with 0.1% Triton X-100, allowingtrypsin to access the cytoplasmic and membrane regionsof WaaL. Under these conditions, the amount of intact

Table 1. Strains and plasmids used in this work.

Strain or plasmid Relevant properties Source or reference

StrainsCLM24 W3110, DwaaL Feldman et al. (2005)DH5a F- p80lacZM15 endA recA hsdR(rK

–mK–) Laboratory Stock

supE thi gyrA relA D(lacZYA-argF) U169SØ874 lacZ trpD(sbcB-rfb) upp rel rpsL Neuhard and Thomassen (1976)SCM3 SØ874, DwaaL This studyW3110 Rph-1 IN(rrnD-rrnE)1 Laboratory Stock

PlasmidspBADFLAG pBAD24 derivative to construct C-terminal fusions to

the FLAG epitope, inducible expression with arabinose, ApRSaldías et al. (2008)

pBADGFP pBAD24 expressing gfpmut3 under the control of arabinose This studypBADHIS pBAD24 derivative to construct C-terminal fusions to the

5xHis epitope, inducible expression with arabinose, ApRLehrer et al. (2007)

pCM234 pBADFLAG, waaLFLAG, ApR This studypCM235 pBADFLAG, waaLFLAG-5xHis, ApR This studypCP20 FLP +, l cI857+, Repts, ApR, CmR l pR Datsenko and Wanner (2000)pFV25 Plasmid encoding GFPmut3A Valdivia et al. (1996)pJHCV32 wc(rfb)Ec07 cosmid, TcR O7+ Valvano and Crosa (1989)pJPD1 pCM235, waaLR265A, ApR This studypJPD2 pCM235, waaLD272A, ApR This studypJPD3 pCM235, waaLG286A, ApR This studypJPD4 pCM235, waaLP303A, ApR This studypJPD5 pCM235, waaLH337A, ApR This studypJPD6 pCM235, waaLG295A, ApR This studypJPD7 pCM235, waaLF299A, ApR This studypJPD8 pCM235, waaLK301A, ApR This studypJPD9 pCM235, waaLH324A, ApR This studypJPD10 pCM235, waaLN325A, ApR This studypJPD11 pCM235, waaLG349A, ApR This studypJPD13 pCM235, waaLR328A, ApR This studypJPD14 pCM235, waaLH335A, ApR This studypJPD15 pCM235, waaLN338A, ApR This studypJPD16 pCM235, waaLR288H, ApR This studypJPD17 pCM235, waaLR288Q, ApR This studypJPD18 pCM235, waaLR288K, ApR This studypJPD19 pCM235, waaLR288E, ApR This studypJPD20 pCM235, waaLR216A, ApR This studypJPD21 pCM235, waaLR216K, ApR This studypJPD22 pCM235, waaLR216H, ApR This studypJPD23 pCM235, waaLR216Q, ApR This studypJPD24 pCM235, waaLR216E, ApR This studypKD46 g, b, and exo from l phage, araC-ParaB, ApR Datsenko and Wanner (2000)pMF19 wbbLEcO16, SpR Feldman et al. (1999)pMM1 pCM234, waaL1-269:340-437FLAG (WaaLDEL5), ApR This studypMM3 pCM235, waaL1-265:GFP, ApR This studypMM4 pCM235, waaL1-419:GFP, ApR This studypMM6 pCM235, waaLR288A, ApR This studypMM12 pCM235, waaLY276A, ApR This study



WaaLFLAG was almost negligible and the 25 kDa cleavageproduct lost (Fig. 4A, lane 1). Also, we did not detect a~3 kDa fragment corresponding to the predicted cleavagesite in EL6 (data not shown). Therefore, trypsin cleavageof WaaL under native conditions is restricted to a limitedamount of accessible cleavage sites in the periplasmyielding fragments containing the C-terminal FLAGepitope. The ~19 kDa C-terminal fragment, also foundafter digestion of Triton X-100-treated samples (Fig. 4A,

lane 1), suggests that cleavage occurs within the EL5. Toconfirm this, spheroplasts were prepared from E. coli cellsexpressing WaaLDEL5 (pMM1) and incubated with trypsinfor 4 h. The Western blot revealed two bands of ~35 and~42 kDa (Fig. 4B, lane 1). The ~35 kDa band likely corre-sponds to a C-terminal fragment from proteolysis occur-ring at any of the two cleavage sites within EL2, while the~42 kDa band represents the undigested WaaLDEL5protein. Cleavage products were absent without trypsin

A

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Fig. 2. Topology of the WaaL protein based on C-terminal GFP fusions. Protein expression was examined by Western blot and fluorescencemicroscopy.A. Western blot analysis using anti-GFP antiserum on total membranes isolated from E. coli K-12 DH5a cells expressing the WaaL–GFPfusion proteins. Lanes: M, Broad Range Prestained SDS-PAGE Standard (Bio-Rad); 1, WaaLGFP (pMM4); 2, GFPmut3 (pBADGFP).B and C. E. coli DH5a cells expressing GFP constructs were immobilized in glass plates coated with 0.8% agarose and imaged byfluorescence microscopy at 1000¥ magnification as described in Experimental procedures. (B) Fluorescence microscopy, WaaLGFP (pMM4).(C) Fluorescence microscopy, GFPmut3 (pBADGFP).

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Fig. 3. Functional analysis of the WaaL protein carrying a C-terminal GFP fusion.A. Surface LPS O antigen in a DwaaL mutant. Silver-stained LPS preparations of E. coli CLM24 carrying pMF19 (for the expression of theO16 LPS) and one of the following plasmids: lane 1, WaaLGFP (pMM4); lane 2, pBADFLAG; lane 3, WaaLDEL5 (pMM1); lane 4, WaaLFLAG

(pCM234). LPS were isolated from cells grown in the presence of 0.2% arabinose, separated on a 14% Tricine-SDS-polyacrylamide gel andsilver stained. O antigen, lipid A-core OS ligated to polymeric O antigen; core + 1, lipid A-core OS plus 1 O unit; core, lipid A-core OS.B. Western blot with anti-FLAG M2 monoclonal antibodies of total membrane preparations that were incubated at 45°C for 30 min. Lanes: M,Broad Range Prestained SDS-PAGE Standard (Bio-Rad); 1, WaaLFLAG (pCM234); 2, WaaLDEL5 (pMM1); 3, pBADFLAG. Asterisks indicate themigration of oligomeric forms of WaaL (see Results).



(Fig. 4B, lanes 2 and 4). Together, the combinedresults of trypsin cleavage experiments of WaaL andWaaLDEL5, and the lack of GFP-mediated fluorescenceof WaaL1-R265-GFP indicate that EL5 is exposed to theperiplasmic space.

Larger bands of variable intensity reacting with the anti-FLAG antibody were observed in all the Western blots(Figs 3B and 4A and B, asterisks). We interpreted thesebands as corresponding to oligomeric forms of WaaL.This is not uncommon in membrane proteins, particularlyin those proteins with high isoelectric points (Kashino,2003). The mild denaturing conditions used for the prepa-ration of the cell lysates, probably not sufficient to dis-perse protein oligomers, could account for the presenceof larger bands, as we have also observed in previousstudies with the integral membrane proteins WecA (Amerand Valvano, 2000; Lehrer et al., 2007), Wzx (Maroldaet al., 2004) and WbaP (Saldías et al., 2008). Thesebands had a variation in mass that was proportional to thevariation found in the respective monomeric proteins(Figs 3B and 4A and B, asterisks), suggesting that thepresence of EL5 does not influence the formation of theoligomeric forms of WaaL.

Identification and functional analysis of conservedamino acids in EL5

To identify conserved residues in EL5 we used theSequencing Alignment and Modelling system (SAM-T02),a suite of automated tools for modelling-, aligning- anddiscriminating-related protein sequences based on alinear hidden Markov model (Karplus et al., 2003). Thisanalysis revealed that Arg-265, Asp-272, Gly-286, Arg-288, Pro-303 and His-337 (the numbers denote the posi-tion of these residues in the E. coli K-12 WaaL) were themost conserved residues in proteins annotated as puta-tive O antigen ligases and/or putative O antigen poly-merases (Fig. 5A and data not shown).

These conserved amino acids were individuallyreplaced by alanine and the ability of each mutant proteinto complement LPS O antigen surface expression in theDwaaL mutant CLM24(pMF19) was evaluated by silverstaining and Western blotting with anti-O16- and anti-coreOS-specific antibodies. LPS from the negative control,strain CLM24(pMF19, pBADFLAG), shows no O antigendetectable by silver staining or anti-core OS antibodies,while a small amount of O16-specific polymeric O antigenwas detected with anti-O16 antibodies (Fig. 5B, arrow).This likely represents Und-PP-linked O16 antigen thatcannot be ligated to lipid A-core OS. In contrast, LPS fromthe positive control, strain CLM24(pMF19, pCM235),forms polymeric O antigen and a band corresponding tocore + 1, both of which are detectable by silver stain,anti-O16 and anti-core OS antibodies (Fig. 5B), indicatingthat WaaL function has been restored.

Mutants R265A, D272A and G286A led to reduced Oantigen surface expression, as evidenced by a reducedamount of polymeric O16 LPS and the lack of a detectableband corresponding to core + 1 (Fig. 5B). Polymeric O16LPS was also detected using anti-core OS antibodies,suggesting that WaaL function is not completely impairedin these mutants. In contrast, the DwaaL strain carryingWaaL mutants H337A and R288A showed an even furtherreduction in polymeric O antigen, and more importantly,polymeric O antigen did not react with the coreOS-specific antibody (Fig. 5B). This indicates that theO16-specific material produced by these mutants, whichis weakly detected by silver stain (especially in the R288Amutant) but detectable with the anti-O16 serum, corre-sponds to Und-PP-linked polymeric O antigen not ligatedto lipid A-core OS. The replacement P303A did not affectWaaL function, as the O antigen was identical to thatproduced by the parental strain (data not shown). Theobserved differences in LPS O antigen surface expres-sion were not due to differences in the protein expressionof the WaaL mutants (data not shown). Also, these differ-ent phenotypes were not due to loading artefacts sincegel loading was normalized according to the bacterial

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Fig. 4. Periplasmic localization of EL5 by protease accessibilityexperiments in spheroplasts. Spheroplasts were treated with trypsinas indicated in each panel and cell lysates examined by Westernblot with anti-FLAG M2 monoclonal antibodies. Asterisks in allpanels indicate the migration of oligomeric forms of WaaL (seeResults). M, Broad Range Prestained SDS-PAGE Standard(Bio-Rad).A. Spheroplasts of bacterial cells expressing WaaLFLAG (pCM234)were incubated with no trypsin for 4 h (0) or with trypsin for 2 h (2)and 4 h (4). Triton X-100 was added (+) prior to incubation withtrypsin. Digested samples were separated on a 16% Tris-GlycineSDS-PAGE. Arrows indicate the migration of the cleavage products.B. Spheroplasts of bacterial cells expressing WaaLFLAG wereincubated with no trypsin (0) or with trypsin (4) for 4 h. Lanes: 1and 2, WaaLDEL5 (pMM1 encoding WaaL1-269/340-437FLAG); 3 and 4,WaaLFLAG (pCM234). Digested samples were separated on a 14%Tris-Glycine SDS-PAGE. Arrows indicate the migration of thecleavage products.



density at the starting point of the LPS preparations (notethat the lipid A-core OS band in the silver-stained gel hasthe same intensity in all lanes; Fig. 5B). Previous work inour laboratory has shown that normalization by bacterialdensity is comparable to other methods including mea-surement of the concentration of 3-deoxy-D-manno-octulosonic acid (a conserved sugar component in thelipid A-core OS) or the amount of protein present in thewhole-cell lysates before treatment with proteinase K(Vinés et al., 2005).

We also investigated the phenotypes mediated by theWaaL mutants in EL5 using the E. coli K-12 strain SCM3carrying pJHCV32 (Table 1), which expresses O7 LPS.O16 and O7 are chemically distinct (L’vov et al., 1984;Stevenson et al., 1994). However, the same results asthose observed with O16 LPS surface expression were

obtained in the presence of WaaL mutants R265A, D272A,G286A, R288A, P303A, H337A (data not shown), indicat-ing that the observed phenotypes are independent of thecomposition and structure of the O antigen. Together, theseexperiments indicate that the conserved EL5 residues canbe separated into three functional groups: residues that arerequired but not essential for full WaaL functionality (Arg-265, Asp-272 and Gly-286), residues absolutely essentialfor function (Arg-288 and His-337) and residues with nofunctional effect on ligation (Pro-303).

A three-dimensional structural model of EL5 predicts aputative ‘catalytic centre’

To our knowledge, there is no structural information onWaaL proteins and also no crystallized structures with

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O antigen

core + 1core

Fig. 5. Functional analysis of conserved amino acids in EL5.A. Representation of conserved amino acids in EL5 obtained by the SAM-T02 protein structure prediction server. The size of the letterrepresenting the amino acid is proportional to the level of conservation of this residue. The position of the most conserved residues isindicated below the letter (R265, D272, G286, R288, P303 and H337), and it was based on the automatic alignment by the server, using 55sequences from putative WaaL homologues of sequenced strains of E. coli, S. flexneri, S. boydii, S. dysenteriae, S. sonnei, Pectobacteriumatrosepticum, Erwinia carotovora, Serratia marcescens, Klebsiella pneumoniae, Chromobacterium violaceum, Citrobacter koseri, Ralstoniametallidurans, R. picketti, R. solanacearum, Vibrio sp., Nitrosomonas europea, Nitrospira multiformis, Haemophilus influenzae, BordetellaPetrii, Cupriavidus taiwanensis, Psychomonas sp., Comamonas testosteroni and Burkholderia phymatum.B. LPS profile of CLM24/pMF19 cells expressing WaaL mutants in EL5 conserved amino acids. Each amino acid was replaced with alanine.Lanes with pBAD and pCM235 correspond to negative and positive controls respectively. LPS samples were run in a 14% Tricine-SDS geland stained with silver (Silver stain), or transferred to nitrocellulose membranes and reacted with polyclonal anti-O16-specific antibodies(Western anti-O16) or monoclonal anti-lipid A-core OS-specific antibodies (Western anti-core OS). O antigen, lipid A-core OS ligated topolymeric O antigen; core + 1, lipid A-core OS plus 1 O unit; core, lipid A-core OS. Arrow indicates polymeric O antigen that does not reactwith anti-core antibodies.



enough similarities to allow protein modelling by threadingor other methods that rely on one or more evolutionaryrelated protein structures as templates. Several second-ary structure prediction algorithms predicted that EL5 ismostly a-helical, and most of the residues that are con-served in the alignments with WaaL homologues fromother bacterial species are also present within predicteda-helices (data not shown). To gain further insight about aputative three-dimensional structural model of EL5, weemployed de novo predictions. In this approach there isno strong dependence on database homology informa-tion, as the structural prediction relies on general prin-ciples governing protein structure and energetics. A denovo model of the EL5 from WaaLEcK12 was generated bythe PROTINFO server of the University of Washington(Hung et al., 2005; 2007) using as input the sequence ofresidues Asn-259 to Lys-347 (Fig. 1). The proposed EL5structural model consists of two pairs of almost perpen-dicular a-helices (Fig. 6). One a-helix from each pair(helices II and IV) contributes to form a structure contain-ing a discrete region that is rich in positively chargedamino acids exposed to the solvent. In particular, themodel shows that most critical EL5 amino acids for ligasefunction, Arg-288 (contributed by a-helix II) and His-337(contributed by a-helix IV), face each other (Fig. 6). Thestructural characteristics of this discrete region suggest itis a putative ‘catalytic centre’.

To evaluate the predictive value of the model, severalalanine replacements were constructed in residues situ-ated in helices II and IV but away from Arg-288 andHis-337 (Gly-295, Phe-299, Lys-301 and Gly-349) andalso in the boundary between helices III and IV (His-324and Asn-325) (Fig. 7A). Alanine replacements of these

residues resulted in mutant proteins that were correctlyexpressed in the membrane (data not shown) and did notaffect WaaL function, as determined by the ability of eachmutant protein to complement O16 LPS surface expres-sion in CLM24(pMF19) (Fig. 7A, silver-stained gel). Addi-tional alanine replacements were made in non-conservedamino acids located at positions surrounding the spatiallocation of Arg-288 and His-337. Tyr-276, Arg-328, His-335 and Asn-338 were targeted in this region becauseof their chemical characteristics (positive chargesand exposed OH groups). None of these replacementsaffected WaaL function, as compared with the parentalprotein (Fig. 7B, silver-stained gel).

The conserved residues that when mutated resulted inreduced WaaL function (Fig. 5B) map to a-helices I (Arg-265 and Asp-272) and II (Gly-286) (Fig. 6). A model of EL5containing the G286A replacement showed that the dis-tance between Arg-288 and His-337 in the mutant proteinwas greater than in the parental EL5 (5.3 Å in the parentalEL5 and 13.6 Å in EL5G286A; Fig. 8). Similar alterations inspacing and angles of Arg-288 relative to His-337 wereobserved in models of EL5 with R265A and D272Areplacements (data not shown). Together, the resultssuggest that these residues contribute to maintain theoverall structure of the putative EL5 ‘catalytic centre’, inparticular the spacing of the ‘cross-over’ region ofa-helices II and IV, defined by the proximity between theside-chains of Arg-288 and his-337.

Predicted structures of E. coli K-12, R1 and R4 ligases

To evaluate the general applicability of the EL5 structuralmodel, we also modelled the large periplasmic loop of

Front view Back view Top view

R288 G286

P303 R265

H337

D272I

II

IIIIV R288

G286

P303

R265

H337

D272

I

II

III

IV

R288

G286

P303

R265

H337

D272I

II

IIIIV

C CC

N

NN

Fig. 6. Structural model of EL5. De novo predicted tertiary structure of E. coli K-12 WaaL EL5 constructed using molecular dynamics (serverPROTINFO). The model has been rotated to facilitate the visualization of the critical residues investigated in this work. Front, back and top viewof two pairs of perpendicularly oriented a-helices. The position of the conserved residues is indicated and coded according to their function(red, critical residues Arg-288 and His-337; blue, Arg-265, Asp-272 and Gly-286 residues that if mutated to alanine result in reduced WaaLfunction; yellow, Pro-303 that if mutated to alanine does not affect WaaL function). N, N-terminus; C, C-terminus. The dotted line points to theC-terminus that is located behind helix I in the back and top views. Note that both the N- and C-terminus are oriented towards the same planesuggesting continuity with the transmembrane segments.



WaaL proteins from R1 and R4 E. coli strains and com-pared them with the WaaLEcK12 model. Despite the lowprimary amino acid sequence similarity among these pro-teins, the models showed a similar structural arrangementand in all of them pairs of perpendicular a-helices werefound (Fig. 9). Also, the conserved arginine residue cor-responding to Arg-288 in WaaLEcK12 is part of a conservedGXR motif that according to the model is exposed to thesolvent. This motif faces a conserved His residue thatcorresponds to His-337, within a conserved HXH motif. Inthe R1 and R4 ligase models the arginine and histidineresidues were also contributed by helices II and IVrespectively (Fig. 9). Highly similar tri-dimensional modelsobtained from dissimilar protein sequences stronglysuggest a conserved tertiary structure in the large peri-plasmic loop of WaaL proteins. Unfortunately, modellingthe predicted periplasmic loops of S. enterica serovarTyphimurium, E. coli R2 and E. coli R3 WaaL proteins

Front view Back view

G295A

K301A

F299A

H324A

G349A

N325A

Y276A

R328A

N338A

H335A

A

B

G295

H324

G349

F299

N325

I

*

G295

F299

G349

H324N325

*

*

R328

Y276

H335

N338

*

R328

Y276

H335

N338

O antigen

core + 1

core

O antigen

core + 1

core

K301

K301

Fig. 7. Functional analysis of non-conserved residues predicted to be near and far from the putative catalytic centre of EL5. The effect ofthese residues in WaaL function was examined by complementation of O16 LPS surface expression as determined by silver staining. Oantigen, lipid A-core OS ligated to polymeric O antigen; core + 1, lipid A-core OS plus 1 O unit; core, lipid A-core OS.A. Front and back views of the EL5 structural model indicating the position of Gly-295, Phe-299, Lys-301, His-324, Asn-325 and Gly-349. All ofthese residues are located opposite to the predicted catalytic centre (asterisk), and were mutated to alanine.B. Front and back views of the EL5 structural model indicating the position of Tyr-276, Arg-328, His-335 and Asn-338. All of these residues arelocated around the predicted catalytic centre (asterisk), but on the other side of helices II and IV, and were mutated to alanine.

5.3 Å(EL5)

13.6 Å(G288A)

EL5

EL5G288A

Fig. 8. Structural alignment of EL5 (green) and EL5 from theWaaLG288A mutant (pink) showing that the His-337 (blue) andArg-288 (red) residues have altered spacing in the mutant protein.



afforded a large content of unstructured regions preclud-ing comparisons among this group of ligases (data notshown).

A positive charge in Arg-288 and in a conservedarginine of the predicted periplasmic loop 4 is requiredfor WaaL activity

Based on its location in the proposed ‘catalytic centre’ andthe strong phenotype of the R288A mutant, Arg-288seems to be a critical amino acid for WaaL function.Together with His-337 (also in EL5) and Arg-216 (in EL4),Arg-288 is the most conserved residue in WaaL proteins(Fig. 5A). To determine whether Arg-288 functions in theligation reaction or is only required for structural purposes,we constructed replacements R288H, R288K, R288E and

R288Q. These mutant proteins led to different levels ofLPS O antigen expression in silver-stained gels and inWestern blots reacted with the anti-O16 antiserum(Fig. 10A). These differences could not be attributed toprotein expression, since all the replacement mutant pro-teins had equal levels of expression and they were allinserted in the plasma membrane (data not shown).WaaLR288K was the only replacement that led to polymericO antigen detectable with the anti-lipid A-core OS antise-rum, suggesting that this mutant was able to restore O16LPS surface expression (Fig. 10A). This result indicatesthat a positive charge in Arg-288 is required for WaaLactivity. However, restoration of O16 LPS expression byWaaLR288K was not complete since as compared withparental WaaL, the amount of polymeric O antigen washighly reduced in bacteria containing the mutant protein.

R4R1K12

H337

R288

I

II

III

IV

C N

R281

H331

I

II IIIIV

N

C

H337

R288

I

III

IV

II

N

C

* * * * * * *** ** * * * *** * *** * ** *WaaL-R4 -KEIDRRINSLKADVISYATKNNSQSSVGARFAMVNAGIKGS-PDG-FNWQSLEQRAEKIKALSAENNIYSGALLFLDVHMHNEIVESLSTKGK 91WaaL-K12 NKPIQNRYNEALNDLNSYTNAN-SVTSLGARLAMYEIGLNIF-IKSPFSFRSAESRAESMNLLVAEHNRLRGALEFSNVHLHNEIIEAGSLK-- 90WaaL-R1 KDTLLMRMNDLNNDLVNYSHDN-TRTSVGARLAMYEVGLKTYSPIG----QSLEKRAEKIHELEEKEPRLSGALPYVDSHLHNDLIDTLSTR-- 87

I II III IV

A

B

Fig. 9. Comparison of structural models of large periplasmic loops of WaaL proteins from E. coli K-12 (EL5), E. coli R1 and E. coli R4.A. The topology of the large periplasmic loops of WaaL proteins of R1 and R4 E. coli was predicted with TMHMM, TOPPRED and HMMTOP. Theamino acid sequences corresponding to the predicted large periplasmic loops were obtained and used for model generation. The a-helices I toIV are indicated, as well as the C-terminal (C) and N-terminal (N) ends of each protein. The models were rotated to facilitate visualization ofthe critical arginine and histidine residues.B. CLUSTAL W alignment of the large periplasmic loops of E. coli K-12, R1 and R4 WaaL proteins. Residues in red correspond to the a-helicesI to IV, as indicated. Asterisks denote conserved residues in the three sequences. Dotted boxes indicate the GXR and HXH motifs in a-helicesII and IV, respectively, containing the critical arginine and histidine residues.



These differences could be attributed to the extendedlength of the aliphatic carbon chain of the lysine residuethat could alter spacing relationships in the ‘catalyticcentre’.

The periplasmic loop 4 (EL4) of E. coli K-12 WaaL ispredicted to consist of three amino acids (Fig. 1), one ofwhich (Arg-216) is also highly conserved in all the ligaseswe have examined (data not shown). We investigated thefunction of this residue by constructing replacementmutants R216A, R216H, R216K, R216Q and R216E.Similar to the R288K replacement, WaaLR216K was the onlyderivative that remained functional as determined by therestoration of polymeric O antigen detectable by silverstaining, an anti-O16 and anti-lipid A-core OS antibody(Fig. 10B and data not shown). This result demonstratesthat a positive charge in Arg-216 is also critical for WaaLfunction.

Discussion

Using a combination of bioinformatics, C-terminal GFPfusions and protease accessibility experiments, we havederived a topological model of the E. coli K-12 WaaL,which consists of 12 predicted transmembrane helicesand has the N- and C-termini oriented towards thecytoplasm. The experimental demonstration that theWaaL C-terminus is located in the cytoplasm agrees withthe results of a genomic-scale topology analysis of innermembrane proteins in E. coli K-12 (Daley et al., 2005).Furthermore, the trypsin cleavage pattern of a WaaLFLAG

derivative after formation of protoplasts supports the

assignment of EL4 and EL5 loops as exposed to theperiplasm. EL5 is also analogous to the periplasmic loopIV of the V. cholerae WaaL (Schild et al., 2005) and the49-amino-acid loop predicted in the WaaL protein fromP. aeruginosa (Abeyrathne and Lam, 2007). A predictedlarge periplasmic loop similar to the EL5 of WaaLEcK12 is acommon feature in other E. coli ligases (Heinrichs et al.,1998a) and also in a large number of putative ligases froma wide range of bacterial species, which were identifiedfrom genomic sequences in public databases (J.M. Pérezand M.A. Valvano, unpublished).

A relatively large periplasmic loop in WaaLEcK12 andother ligases strongly suggests that this loop has a func-tional role, which is consistent with the periplasmic loca-tion of the ligation reaction (Mulford and Osborn, 1983).Thus, it is not surprising that deletion of EL5 leads to theloss of WaaLEcK12 function. Comparative analyses withavailable sequences identified six residues in EL5 that arestrongly conserved. From these, Arg-288 and His-337showed the highest conservation. WaaL mutants withalanine replacements at any of these two positionsshowed the most dramatic effect in ligase function. In themajority of WaaL sequences from experimentally anno-tated ligases, and also in most WaaL proteins identified ingenome sequences of many Gram-negative bacteria,residues corresponding to Arg-288 and His-337 are withinconserved GXR and HXH motifs (J.M. Pérez and M.A.Valvano, unpublished). Other ligases lacking the GXRand HXH motifs (such as the WaaL proteins fromS. Typhimurium, S. Arizonae, S. Diarizonae, E. coli R2,E. coli R3 and Shigella flexneri ) have SSYRY and

Fig. 10. Functional role of positive chargesin positions 288 and 216 in WaaL activity.LPS samples from CLM24/pMF19 cellsexpressing WaaL and WaaL mutants were runin a 14% Tricine-SDS gel and stained withsilver (Silver stain), or transferred tonitrocellulose membranes and reacted withanti-O16-specific antibodies (Westernanti-O16) or anti-lipid A-core OS-specificantibodies (Western anti-core OS). O antigen,lipid A-core OS ligated to polymeric Oantigen; core + 1, lipid A-core OS plus 1 Ounit; core, lipid A-core OS. Arrows indicatepolymeric O antigen that does not react withanti-core antibodies.A. pBAD, WaaL (pCM235), and the WaaLmutants R288Q, R288K, R288H or R288E.B. pBAD, WaaL (pCM235), and the WaaLmutants R216Q, R216K, R216H or R216E.

pBAD

pCM

235

R216Q

R216K

R216H

R216E

pBAD

pCM

235

R216Q

R216K

R216H

R216E

pBAD

pCM

235

R216Q

R216K

R216H

R216E

Western anti-O16 Western anti-core OSSilver stain

pBAD

pCM

235

R288Q

R288K

R288H

R288E

pBAD

pCM

235

R288Q

R288K

R288H

R288E

pBAD

pCM

235

R288Q

R288K

R288H

R288E

Western anti-O16 Western anti-core OSSilver stain

A

B

O antigen

core + 1

core

O antigen

core + 1

core



SIQPHN motifs at equivalent positions, which also containconserved arginine and histidine residues (underlined)respectively. These observations underscore the evolu-tionary conservation of the critical arginine and histidineresidues in diverse WaaL proteins, suggesting theseamino acids may play key roles in the ligation reaction.

The loss or reduction in WaaL function in amino acidreplacement mutants of the conserved residues in EL5might be caused by a defective interaction of theenzyme with either Und-PP-linked substrates or the lipidA-core OS acceptor. It has been proposed that since aspecific ligase can ligate distinct O antigens to the samelipid A-core molecules, the structure of the terminal coreOS determines ligase specificity (Heinrichs et al.,1998a,c). Our results showing that the WaaL mutantscaused the same LPS O antigen phenotypes with theO16 and O7 systems agree with this model and suggestit would be unlikely that the conserved residues of EL5participate in recognition of core OS terminal sugars.Recently, it was reported that purified WaaL of P. aerugi-nosa possesses ATPase activity and ATP hydrolysis isrequired for the ligation reaction in vitro (Abeyrathne andLam, 2007). Unfortunately, well-conserved motifs forATP binding and/or hydrolysis are not apparent in theP. aeruginosa WaaL or in other WaaL proteins. Ourefforts to mutate amino acids of regions in the E. coliK-12 WaaL showing a low level of similarity with WalkerA and B motifs (Walker et al., 1982) and a palmate motif(Yamaguchi et al., 1993) did not result in any mutantprotein with compromised WaaL function that could beattributable to ATP hydrolysis (Fig. S1). In any case, itwould be unlikely that the conserved residues in EL5 areinvolved in ATP hydrolysis since ATP does not occur inthe periplasm (Pugsley, 1993). Therefore, we proposethat the conserved EL5 residues, which are commonacross WaaL proteins with different lipid A-core OSspecificities, participate in the recognition of Und-PP-linked O antigen, and more specifically the Und-PPmoiety, as this is the only common part in the substratemolecules used by all WaaL proteins.

Despite that at least one ligase protein has been suc-cessfully purified (Abeyrathne and Lam, 2007), structuraldata on these proteins are lacking. Furthermore, the lowconservation in the amino acid sequence precludesstructural predictions of the entire WaaL or subregions ofthe protein by comparative modelling and classical foldrecognition methods. However, it is not unusual thatperiplasmic soluble regions between TM helices arestructured (Lee et al., 2008; Parsons et al., 2008; Tociljet al., 2008). Preliminary experiments to purify EL5 inquantities suited for structural analyses resulted ininsoluble protein. Therefore, we reasoned that a molecu-lar dynamics strategy (or de novo modelling) combinedwith mutagenesis could help establish a structural model

of the EL5 that could serve as a template to comparealso with other WaaL proteins. De novo modelling is par-ticularly strong for short peptides of less than 100 aminoacids (Samudrala and Levitt, 2002), and can in principlebe applied to recognize structural folds in solvent-exposed regions of membrane proteins. Our predictedtri-dimensional model of EL5 consists of two pairs ofalmost perpendicular a-helices. Comparisons with otherproteins revealed low-level similarity between the pre-dicted structure of EL5 and that of the catalytic domainsin the ribose 5′-phosphate isomerase (Zhang et al.,2003; Graille et al., 2005) and erythromycin polyketidesynthase (Broadhurst et al., 2003). As predicted for EL5,these enzymes bind phosphorylated molecules as sub-strates or cofactors, respectively, and two pairs ofa-helices arranged similarly to those in EL5 contribute tothe catalytic domains.

The highly conserved amino acids found in WaaL pro-teins, which led to a defective enzyme when replaced inthe E. coli K-12 protein, are distributed within definedregion of EL5. In particular, Arg-288 and His-337 arelocated facing each other. The orientation and the dis-tance between Arg-288 and His-337 appear to be impor-tant for WaaL function. Indeed, according to our model,alanine replacements of neighbouring residues of theGXR motif affect the spatial orientation and distance ofArg-288 relative to His-337, while the global structure ofthe rest of the loop is not altered. This could explain thedecreased ability of these WaaL mutants to complementO antigen synthesis. Also, a positive charge at position288 is critical for WaaL function. From the various replace-ments at this position, only the R288K did not affect sig-nificantly ligase function while other replacementsresulted in a non-functional protein. Thus, we propose thatthe region containing the highly conserved residues ina-helices II and IV defines a putative catalytic reactioncentre. Mutations reported in the literature in the con-served histidine residues of the periplasmic loops of WaaLfrom P. aeruginosa and V. cholerae (both correspondingto His-337) also affected ligase function in these systems(Schild et al., 2005; Abeyrathne and Lam, 2007), support-ing even further the notion that this residue is critical forthe activity of different ligases. As predicted from ourmodel, additional alanine replacements of non-conservedEL5 residues both near and outside the putative catalyticregion resulted in mutant WaaL forms that remainedfunctional. Similarly, mutations made in non-conservedresidues in the predicted periplasmic loop of theP. aeruginosa WaaL did not affect ligase function(Abeyrathne and Lam, 2007).

WaaL proteins show significant divergence in theirprimary amino acid sequence, even for members fromthe same species, but in contrast, hydrophobicity plotanalyses show predictable secondary structural features



(Heinrichs et al., 1998a; Nesper et al., 2002). Forinstance, in E. coli there are five chemically distincttypes of lipid A-core OS (K-12, and R1 to R4) andstrains with these types also have different WaaL pro-teins. We modelled the structure of the EL5 counterpartsof WaaL proteins from E. coli R1 and R4 types. Remark-ably, despite the divergence in the amino acidsequences of EL5-like loops the three models resultedin highly similar structures all consisting of foura-helices. Virtually all conserved amino acids werelocated in the same putative catalytic region and moreimportantly the critical residues at positions equivalent toArg-288 and His-337 of WaaLEcK12, defined by the motifsGXR and HXH, are also placed facing each other.Therefore, our data support a conserved tertiary struc-ture in the large periplasmic loop of the majority of WaaLproteins containing GXR and HXH motifs.

Our study also shows that a highly conserved arginineresidue (Arg-216 in WaaLEcK12) in the short periplasmicloop preceding the large loop also has significant func-tional relevance. An arginine at an equivalent position inthe V. cholerae WaaL was shown to be important for activ-ity (Schild et al., 2005), and we demonstrate here thatArg-216 is required for WaaLEcK12 function. Moreover,similar to Arg-288, replacing Arg-216 with alanine, histi-dine, glutamine and aspartate resulted in non-functionalproteins, while the R216K mutant was able to produceLPS O antigen.

In summary, the results of this study lead us to proposethat key EL5 and EL4 residues of WaaL may be part of acatalytic reaction centre and possibly involved in thebinding of the phosphate groups of Und-PP. Indeed, his-tidine and lysine are critical active-site residues in otherenzyme reactions resolving phosphodiester bonds, wherethese or similar residues act as proton donors or protonacceptors targeting the oxygen atom of one of the phos-phate molecules. Similarly, the binding of ATP phosphategroups in the Walker A domains (R/KxxxxxGxxxL/VhhhD)is mediated by either lysine or arginine residues (Walkeret al., 1982). Further experiments are in progress in ourlaboratory to explore whether or not Arg-288 and Arg-216act in protonation/deprotonation on the phosphate bondsof the Und-PP-linked O antigens. The identification of aconserved structural model for the critical motifs found inWaaL and its corresponding periplasmic location providesa framework to dissect the WaaL reaction mechanism.Given the conservation of this structural motif, and thespecific location of the uniquely conserved amino acids inthe EL5 loop and in similar loops of many other WaaLproteins, we propose that these motifs are not involved inthe specific recognition of the lipid A core acceptor mol-ecules but rather participate in the chemical reaction orreactions required for the release of O antigen from theUnd-PP lipid carrier.

Experimental procedures

Bacterial strains, plasmids and growth conditions

The plasmids and bacterial strains used in this study arelisted in Table 1. Additional plasmids are listed in Table S1.Strain CLM24, an E. coli W3110 DwaaL, was used to assessprotein expression of all constructs and for in vivo comple-mentation studies. Bacteria were cultured at 37°C in Luria–Bertani (LB) medium supplemented with ampicillin(100 mg ml-1), tetracycline (20 mg ml-1) and 0.2% (w/v) arabi-nose, when appropriate. Transformation was performed byeither the calcium chloride method or electroporation. Allbiochemical reagents were purchased from Sigma (St Louis,MO), unless indicated otherwise. Restriction endonucleases,T4 DNA ligase and associated buffers were purchased fromRoche Molecular Biochemicals (Dorval, Quebec, Canada).Broad Range Prestained SDS-PAGE Standard (Bio-Rad)consisted of: myosin (206 kDa), b-galactosidase (119 kDa),bovine serum albumin (91 kDa), ovalbumin (51 kDa), car-bonic anhydrase (38 kDa), soybean trypsin inhibitor (29 kDa)and lysozyme (19 kDa).

Construction of strains and plasmids

For complementation experiments using the LPS O7 systemwe constructed SCM3, a derivative of the E. coli strainSØ874 carrying a deletion of the waaL gene. The parentalstrain carries a large deletion that eliminates the E. coli K-12O16 antigen biosynthesis cluster (Neuhard and Thomassen,1976). This was performed as described by Datsenko andWanner (2000). Primers corresponding to regions adjacentwaaL (5′-GCAGTTTTGGAAAAGTTATCATCATTATAAAGGTAAAACATGTGTAGGCTGGAGCTGCTTCG and 5′-AGTGAGTTTTAACTCACTTCTTAAACTTGTTTATTCTTAACATATGAATATCCTCCTTAG) contained 20 additional nucleotidesthat annealed to the template DNA from plasmid pKD4 (initalics), which carries a kanamycin resistance gene flankedby FRT (FLP recognition target) sites. Competent cells wereprepared by growing SØ874 carrying pKD46 (Datsenko andWanner, 2000) in LB containing 0.5% (w/v) arabinose, andthe PCR products were introduced by electroporation. Theplasmid pKD46 encodes the Red recombinase of the phage,which was placed under the control of the arabinose-inducible promoter PBAD. Kanamycin-resistant colonies werescreened by PCR with primers annealing to regions outsideof the mutated gene. Next, the antibiotic gene was excised byintroducing the plasmid pCP20 (Datsenko and Wanner, 2000)encoding the FLP recombinase. Plasmids pKD46 and pCP20are both thermosensitive for replication and were cured at42°C. The resulting strain was transformed with cosmidpJHCV32 carrying the genes encoding O7 LPS (Valvano andCrosa, 1989). Plasmid pCM234, encoding WaaL C-terminallyfused to the Flag epitope (WaaLFLAG), was constructed byPCR amplification of a 1.27 kb fragment encoding waaL fromE. coli strain W3110 with primers containing restriction sites(underlined) EcoRI (5′-GACGGAATTCATGCTAACATCCTTTAAACTTC-3′) and SmaI (5′-CGTCCCGGGATTAATTGTATTGTTACG-3′). The PCR product was ligated (Rapid Liga-tion Kit, Roche Diagnostics) to pBADFLAG also digested withEcoRI and SmaI. Plasmid pCM235, encoding the WaaLFLAG



C-terminally fused to a 5xHis tag, was constructed by ampli-fication of a 732 bp fragment using pCM234 as a DNAtemplate and vector primer (5′-GATTAGCGGATCCTACCTGA-3′) and XhoI primer (5′-CGTCCTCGAGCTTGTCGTCGTCGTCGTC-3′) restriction sites (underlined). The PCRproduct and the plasmid pBADHIS were digested with EcoRIand XhoI. To construct plasmid pMM1 encoding a deletion ofthe WaaL external loop 5 (WaaLDEL5) a 5.56 kb PCRproduct was amplified with pCM234 as DNA template andprimers XhoI (5′-CATGCTCGAGATTCTGTATTGGTTTATT-3′) and XhoI (5′-GACTCTCGAGCATCTACATAATGAGATA-3′), digested with XhoI and self-ligated. Plasmid pBADGFPwas created to construct C-terminal GFP fusions. A ~0.9 kbfragment encoding GFPmut3A was amplified from plasmidpFV25 using primers SmaI (5′-GACTCCCGGGAGTAAAGGAGAAGAACTT-3′) and PstI (5′-CATTAAAGCTTGCATGCCTGCAGG-3′). The PCR product and pBAD24 weredigested with SmaI and PstI. Two plasmids carryingWaaLGFP fusion proteins were constructed: pMM3 (encodingWaaL1-265:GFP) and pMM4 (encoding WaaL1-419:GFP). In bothcases the fragments were amplified from pCM234 DNA. ThePCR products and pBADGFP were digested with EcoRI andSmaI. In all the cloning experiments the DNA fragments wereligated with T4 DNA ligase, and the ligation mix introducedinto E. coli DH5a competent cells by transformation. Trans-formants were selected in media containing ampicillin and theappropriate plasmids were sequenced (York University CoreMolecular Biology and DNA Sequencing Facility).

Site-directed mutagenesis

For site-directed mutagenesis of the EL5 of E. coli K-12WaaL, oligonucleotide primers were designed to create therequired amino acid change. The sequences of all primersused for mutagenesis are available from the authors uponrequest. The plasmid pCM235 was used as DNA template.PCR products amplified with Pfu polymerase were digestedovernight with 1 U DpnI at 37°C. The digested PCR productswere introduced into DH5a competent cells by transforma-tion, and transformants recovered by plating onto LB agarplates supplemented with ampicillin. Plasmids were recov-ered and the insert DNA was sequenced to confirm the intro-duction of the correct base-pair change encoding the aminoacid substitution.

LPS and protein analysis

LPS was prepared from cells grown on LB plates with 0.2%(w/v) arabinose as previously described (Marolda et al.,2006). Samples were separated on 14% (w/v) Tricine-SDS-PAGE and the gels stained with silver nitrate (Marolda et al.,2006). Total membranes were prepared from cells grown inLB medium and WaaL expression was induced with 0.2%arabinose. Bacterial cells were suspended in 20 mM Na2PO4

pH 7.4 plus protease inhibitors (Complete Tablets, RocheDiagnostics) and they were lysed by sonic disruption for two15 s pulses (Branson). Total membrane fractions wereobtained by centrifugation of the lysates for 40 min at40 000 g and the pellet re-suspended in the same buffer.Protein concentration was measured by the Bradford assay

(Bio-Rad Protein Assay). Total membrane proteins werere-suspended, mixed with the appropriate amount of loadingsample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glyc-erol and 0.1% bromophenol blue), and incubated at 45°C for30 min before loading. After separation on a SDS-PAGE,membrane proteins were transferred to nitrocellulose mem-branes for immunoblot analysis.

Western blots

Nitrocellulose membranes were blocked with 10% WesternBlocking Solution (Roche diagnostics), followed by three10 min washes with 50 ml of TBS pH 7.5 (50 mM Tris-HCL,150 mM NaCl). Membranes were incubated for 2 h with theprimary antibody: 7 mg ml-1 FLAG M2 monoclonal antibody(Sigma), 1:1000 dilution of O16 polyclonal antiserum, 1:5000dilution of anti-core LPS monoclonal antibody (HyCult bio-technology b.v.), or 1:1000 dilution of anti-GFP polyclonalantibodies (Chemicon International), as appropriate. Thereacting bands were detected by fluorescence with anOdyssey infrared imaging system (Li-cor Biosciences) usingIRDye800CW affinity-purified anti-rabbit IgG antibodies(Rockland, Pennsylvania) and Alexa Flour® 680 anti-mouseIgG antibodies (Invitrogen).

Fluorescence microscopy

Five millilitres of fresh LB containing the appropriate antibioticwas inoculated to an OD of 0.15 from an overnight bacterialculture. Once bacteria reached mid-log phase, WaaL proteinexpression was induced with 0.02% (w/v) arabinose for 3 h.Bacteria were incubated on ice for 1 h to allow sufficient timefor GFP to fold properly. Subsequently, 10–12 ml of culturewas transferred to a microscope slide coated with 0.8%agarose to slow down bacterial movement. Fluorescence andphase-contrast images were obtained using a Qimaging(Burnaby, British Columbia, Canada) cooled charged-coupled device camera on an Axioscope 2 microscope (CarlZeiss, Thornwood, NY). Live images were digitally processedwith the Northern Eclipse version 6.0 imaging software(Empix Imaging, Mississauga, Ontario, Canada).

Isolation of spheroplasts

For spheroplasts isolation bacteria were freshly grown to anOD of 0.7. At this point protein expression was induced with0.2% arabinose and incubated for 2 h at 37°C. Cultures wereplaced on ice for 20 min, and 3 ml aliquots were centrifugedfor 1 min at 16 100 g and the pellet re-suspended in 1 ml ofan 18% sucrose solution in 0.001 mM Tris pH 8. Cell densitywas standardized to an OD600 of 2.0 in a 1 ml solution con-taining 18% (w/v) sucrose, 0.001 mM Tris pH 8, 10 mM EDTAand 100 mg ml-1 lysozyme. The suspension was incubated onice for 30 min and the spheroplasts formation was monitoredby light microscopy. Spheroplasts were collected by centrifu-gation (1 min at 16 100 g) and re-suspended in 70 ml of 18%sucrose in 0.001 mM Tris pH 8.0.

Trypsin digestion

Two microlitres of 1 mg ml-1 Trypsin Sequencing grade(Roche) was added to 70 ml of spheroplasts suspensions and



incubated from 2 to 4 h at 30°C. Trypsin was inhibited with theaddition of 0.1% Trifluor Acetic acid (TFA) and 1¥ protein dye.Total lysate of spheroplasts was obtained with 0.1% TritonX-100. Digested products were identified by immunoblottingwith FLAG M2 monoclonal antibody, as described above.

Computer techniques

Vector NTI suite 7.0 software package (Informax, Bethesda,MD) was used for DNA sequence analysis and oligonucle-otide primer design. CLUSTAL W was used for sequencealignments. BLAST searches were carried out using the toolsprovided by the national centre for biotechnology information.Hydrophobic patterns and topology predictions were per-formed using MEMSAT, PHD, TMHMM, TOPPRED and HMMTOP

servers. For amino acids conservation studies and secondarystructure predictions the HMM-based protein structure pre-diction server SAM TO2 was used (Karplus et al., 2003) withthe input peptide sequence from spanning N259-K357(Fig. 1). Molecular models of WaaL EL5 were constructedusing molecular dynamics techniques provided by thePROTINFO server (http://protinfo.compbio.washington.edu) ofthe University of Washington. Structure visualization andelectrochemical profiles were performed using the programPymol Molecular Graphics System (Delano Scientific LLC;http://pymol.sourceforge.net).

Acknowledgements

We thank M.S. Saldías, K. Patel, D.E. Heinrichs and C.Creuzenet for critical reading of the manuscript, and C.deLasa for constructing pBADGFP and WaaL–GFP. Thisstudy was supported by a grant from the Canadian Institutesof Health Research. M.A.M. was supported in part by anOntario Graduate Scholarship. M.A.V. holds a CanadaResearch Chair in Infectious Diseases and MicrobialPathogenesis.

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functional analysis of the large periplasmic loop of the escherichia coli

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