Molecular Microbiology (2008) 70(6), 14241440

doi:10.1111/j.1365-2958.2008.06490.x First published online 29 October 2008

Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase
Jos M. Prez, Megan A. McGarry, Cristina L. Marolda and Miguel A. Valvano* Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, N6A 5C1, Canada. Nikaido, 1976; Nikaido, 2003). LPS is key to the effective permeability barrier properties of the outer membrane (reviewed in Nikaido, 2003) and consists of lipid A, core oligosaccharide (OS) and the O polysaccharide or O antigen (reviewed in Raetz and Whiteld, 2002; Valvano, 2003). Prior to ligation, the initial synthesis of lipid A-core OS and O antigen polymers occurs independently at the cytoplasmic side of the inner membrane (Raetz and Whiteld, 2002; Valvano, 2003). The lipid A-core OS is translocated to the periplasmic site of the membrane by a process that requires ATP hydrolysis, mediated by the ABC transporter MsbA (Doerrler et al., 2001; Doerrler and Raetz, 2002). The O antigen is synthesized as an undecaprenyl-diphosphate (Und-PP)-linked intermediate, and translocated across the membrane by the Wzy-, ABC transporter- or synthase-dependent pathways (Raetz and Whiteld, 2002). The Wzy-dependent pathway requires the membrane proteins Wzx (Und-PP-O antigen ippase), Wzy (O antigen polymerase) and Wzz (regulator of O antigen polysaccharide chain length distribution) (Raetz and Whiteld, 2002; Valvano, 2003). The ABC transporter-dependent pathway employs the proteins Wzm and Wzt, which are the permease and ATP hydrolysis components of an ABC-class 2 transporter respectively (Raetz and Whiteld, 2002). The synthasedependent pathway occurs in Salmonella enterica serovar Borreze (Keenleyside and Whiteld, 1996) and requires a single protein of the hyaluronan synthase family (Raetz and Whiteld, 2002). Irrespective of the export and polymerization modes of the saccharide molecules, nascent Und-PP-linked O antigens are ligated to terminal sugar residues of the lipid A-core OS (McGrath and Osborn, 1991) in a reaction mediated by the membrane protein WaaL. Mutations in the waaL gene prevent the ligation of O antigen molecules to lipid A-core OS, resulting in bacteria producing a rough LPS (lacking O antigen polysaccharide) and accumulating intracellular Und-PP-linked O antigen molecules (Mulford and Osborn, 1983; McGrath and Osborn, 1991). Since O antigens of pathogenic bacteria are usually required for resistance to complement-mediated killing (Pluschke and Achtman, 1984; Joiner, 1988), the elucidation of the ligase reaction could potentially be exploited for the discovery of novel LPS biosynthesis inhibitors. However, the mechanism of ligation is still unresolved. Although the ligase

Summary
WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic 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 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular a-helices. One a-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.

Introduction
Gram-negative bacteria have an asymmetric outer membrane bilayer rich in phospholipids on the inner leaet and lipopolysaccharide (LPS) on the outer leaet (Kamio and
Accepted 1 October, 2008. *For correspondence. E-mail mvalvano@ uwo.ca; Tel. (+1) 519 661 3427; Fax (+1) 519 661 3499.

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd

Functional analysis of the WaaL large periplasmic loop 1425

catalyses the formation of a glycosidic bond, WaaL proteins bear no relationship with classical glycosyltransferases, which modify sugar nucleotide substrates. The ligation reaction shows no specicity for the type of O antigen or the mechanism of O antigen export (Raetz and Whiteld, 2002). In contrast, a requirement for a specic lipid A-core OS acceptor structure has been established in several models (Heinrichs et al., 1998a,b; Abeyrathne et al., 2005; Schild et al., 2005), which has led to the generalized notion that a specic WaaL protein recognizes a given core OS terminal structure. However, it is unclear how WaaL recognizes the Und-PP-linked O antigen and, in particular, which part of this molecule participates in the enzymatic reaction. WaaL proteins show signicant divergence in their primary amino acid sequence, even for members from the same species (Raetz et al., 2007). The poor sequence conservation among O antigen ligases makes comparative analyses difficult. It is also difficult to establish relationships in WaaL proteins based on potential core OS acceptor structures. For example, the Escherichia coli R2 and S. enterica WaaL proteins share ~80% amino acid sequence similarity and are functionally interchangeable, as both link the O antigen polysaccharide to a terminal glucose in the core OS that has an a-1,2-linked N-acetylglucosamine (Heinrichs et al., 1998a). In contrast, E. coli R3 WaaL is ~66% similar to the Salmonella protein but links the O antigen polysaccharide to a different site of attachment in the core OS that resembles a similar site in the K-12 core OS. However, the R3 WaaL shares very little identity with the E. coli K-12 ligase (Heinrichs et al., 1998c). More recent evidence suggests that the specicity of the ligation reaction for a particular core lipid A-core OS structure does not solely depend on the WaaL protein, but presumed additional factor or factors have not been identied (Kaniuk et al., 2004). Conceivably, WaaL activity requires amino acids exposed to the periplasmic space where they could interact with donor and acceptor molecules. A critical His residue was identied in a periplasmic loop of the Vibrio cholerae WaaL (Schild et al., 2005), and a potentially common motif is emerging not only in WaaL proteins but also in proteins that ligate Und-PP-linked O antigen precursors to pili (Qutyan et al., 2007). Recently, Abeyrathne and Lam (2007) reported that highly puried WaaL from Pseudomonas aeruginosa has ATPase activity and ATP hydrolysis is required for the in vitro ligation reaction. This is an intriguing nding since ATP is not present in the periplasmic space (Pugsley, 1993). In this work, we established a topological model of the E. coli K-12 WaaL ligase and identied highly conserved residues in two adjacent periplasmic loops, which are required for function. Using molecular dynamics, structural models of one of these loops were constructed for

the E. coli K-12, R1 and R4 WaaL proteins. All models revealed a similar fold consisting of two pairs of almost perpendicular a-helices. Highly conserved histidine and arginine residues, required for WaaL function, are located facing each other in the cross-over region between one a-helix of each pair both. Our results provide a predictive model to probe structurefunction relationships in WaaL proteins and lead us to propose the existence of a catalytic region that could interact with Und-PP, the common molecule in the Und-PP-linked O-antigen substrates for the ligation reaction.

Results
Predicted topology of E. coli K-12 WaaL Using current algorithms for predicting transmembrane helices and inout topologies of the intervening loops (Nilsson et al., 2000; Drew et al., 2002), we constructed a topological model for the E. coli K-12 WaaL protein (419 amino acids) that consists of 12 predicted transmembrane domains, six periplasmic loops, ve cytoplasmic loops, and has the N- and C-termini in the cytoplasm (Fig. 1). We investigated the orientation of the WaaL C-terminus using the green uorescent protein (GFP) as a topology probe, since GFP can only stably fold into a chromophore when present in the cytoplasm (Feilmeier et al., 2000; Drew et al., 2002). E. coli DH5a transformants containing pMM4 (which encodes WaaL C-terminally fused to GFP at amino acid 419; WaaLGFP; Table 1) expressed a polypeptide with an apparent mass of 70 kDa, in agreement to the predicted molecular mass of the WaaLGFP fusion protein (Fig. 2A, lane 1), and exhibited green uorescence localized to the periphery of each cell (Fig. 2B). Additional bands of lower molecular mass reacting with the anti-GFP M2 monoclonal antibody (Fig. 2A, lane 1) were interpreted as degradation products. In the control experiment, cells containing pBADGFP expressed a ~30 kDa polypeptide (Fig. 2A, lane 2), similar to the approximate molecular mass of GFPmut3 (27 kDa), and displayed green uorescence uniformly distributed throughout the cell body (Fig. 2C). We conclude from this experiment that the WaaL C-terminus resides in the cytosol. The functionality of WaaLGFP was assessed using the waaL-decient strain CLM24(pMF19) (Feldman et al., 2005). Plasmid pMF19 encodes a rhamnosyltransferase that allows for the completion of the O16 subunit synthesis in E. coli K-12 strains of the W3110 lineage (Liu and Reeves, 1994; Feldman et al., 1999). LPS samples prepared from CLM24(pMF19) containing pMM4 revealed the classical ladder-like pattern of O16 antigen polysaccharide, with lower bands corresponding to lipid A-core OS (core) and lipid A-core OS plus one O unit (core + 1), and higher molecular weight bands corresponding to lipid

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd, Molecular Microbiology, 70, 14241440

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

I GL I K S P NI F F S F R S A ES

A R

YE LA M

303

G
L S T V S N A N T Y S N L

288

R A E

286

EL5
272
A E G E F L A

periplasm

EL4

M N L L V A E H N R L R

D N L

N S P D V F A I I MS FF C L I YT II E L I M M L T S F K L HS K L P S S K L T Y N T F S IK IY N I T AI VC L L S L I L R G R Q E N Y D V K F A S Y W I LD LL G I L FI SL P L I L N K I N F R A T Y H SY LN T A K IF IF G S F IV FL T L T S Q L K

D R I N E H I S N I Y M AY GA I L F SL SY L T Y L V S E K S F G V G T AT GA A Y S TM LI G I V SG VA I L Y T K K Q T L A L VY LV A C S NL LF L F P H N

Y R

265
N Q

337
I P K N F

H LH

R 216
A T L LL FP I I C VA AL I A Y Y N K S

25

I I V I SA LI A I L LV IS S T F K K P

NH2

N A I E I 347 W E A G I I L S V D S K G L L GI M G GV I FS I T L G F L TL L Y IL FS LF L G L Y I A A Y R K K

R S I P II II S A I VL LL V I N N R N N T I N

S K

419
COOH

cytoplasm

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 function respectively (except for P303, see Results). Thick bars indicate the end-points of the deleted region in WaaLDEL5 (pMM1).

A-core OS ligated to polymeric O antigen (Fig. 3A, lane 1). A similar result was obtained with CLM24(pMF19) containing pCM234 (expressing WaaL with a C-terminal FLAG fusion; Fig. 3A, lane 4) or pCM235 (expressing WaaL with a C-terminal FLAG-5xHis fusion; data not shown). The negative control strain containing the pBADFLAG cloning vector did not produce LPS O antigen (Fig. 3A, lane 2). Therefore, the WaaL C-terminus resides in the cytosol and can also tolerate protein fusions without a noticeable effect in WaaL function. The external loop 5 is required for WaaL function and faces the periplasm All WaaL homologues have a predicted large periplasmic loop of variable length (Schild et al., 2005; Abeyrathne and Lam, 2007; J.M. Prez 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 O antigen synthesis, we constructed pMM1. This plasmid encodes a deletion derivative of WaaL (WaaLDEL5) that lacks EL5 and also 5 amino acids from the TM10 (Asn-

259 to Lys-347, Fig. 1). The last 5 amino acids of TM10 were included in the deletion since the precise EL5TM10 boundary was not certain and lysine residues are not typical in TM helices. CLM24(pMF19, pMM1) did not produce LPS O antigen (Fig. 3A, lane 3), despite that the protein was expressed and correctly localized to the membrane (Fig. 3B, lane 2). Therefore, loss of the large periplasmic loop does not affect membrane localization and/or WaaL expression, but an intact loop is required for ligation. To experimentally address the location of EL5, we constructed a C-terminal GFP fusion after Arg-265 (Fig. 1). Although the WaaL1-R265-GFP derivative was detected by Western blotting, E. coli DH5a expressing WaaL1-R265-GFP (pMM3) did not exhibit green uorescence (data not shown), suggesting that the fused GFP moiety resides in the periplasm. The periplasmic location of EL5 was also investigated in the full-length WaaL by a protease protection assay using intact spheroplasts of E. coli cells expressing WaaLFLAG (pCM234). Spheroplasts are useful for membrane topology studies since the disruption of the outer membrane exposes the periplasmic domains of plasma membrane proteins, which become accessible to

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Functional analysis of the WaaL large periplasmic loop 1427

Table 1. Strains and plasmids used in this work.

Strain or plasmid Strains CLM24 DH5a S874 SCM3 W3110 Plasmids pBADFLAG pBADGFP pBADHIS pCM234 pCM235 pCP20 pFV25 pJHCV32 pJPD1 pJPD2 pJPD3 pJPD4 pJPD5 pJPD6 pJPD7 pJPD8 pJPD9 pJPD10 pJPD11 pJPD13 pJPD14 pJPD15 pJPD16 pJPD17 pJPD18 pJPD19 pJPD20 pJPD21 pJPD22 pJPD23 pJPD24 pKD46 pMF19 pMM1 pMM3 pMM4 pMM6 pMM12 Relevant properties W3110, DwaaL F- p80lacZM15 endA recA hsdR(rKmK) supE thi gyrA relA D(lacZYA-argF) U169 lacZ trpD(sbcB-rfb) upp rel rpsL S874, DwaaL Rph-1 IN(rrnD-rrnE)1 pBAD24 derivative to construct C-terminal fusions to the FLAG epitope, inducible expression with arabinose, ApR pBAD24 expressing gfpmut3 under the control of arabinose pBAD24 derivative to construct C-terminal fusions to the 5xHis epitope, inducible expression with arabinose, ApR pBADFLAG, waaLFLAG, ApR pBADFLAG, waaLFLAG-5xHis, ApR FLP +, l cI857+, Repts, ApR, CmR l pR Plasmid encoding GFPmut3A wc(rfb)Ec07 cosmid, TcR O7+ pCM235, waaLR265A, ApR pCM235, waaLD272A, ApR pCM235, waaLG286A, ApR pCM235, waaLP303A, ApR pCM235, waaLH337A, ApR pCM235, waaLG295A, ApR pCM235, waaLF299A, ApR pCM235, waaLK301A, ApR pCM235, waaLH324A, ApR pCM235, waaLN325A, ApR pCM235, waaLG349A, ApR pCM235, waaLR328A, ApR pCM235, waaLH335A, ApR pCM235, waaLN338A, ApR pCM235, waaLR288H, ApR pCM235, waaLR288Q, ApR pCM235, waaLR288K, ApR pCM235, waaLR288E, ApR pCM235, waaLR216A, ApR pCM235, waaLR216K, ApR pCM235, waaLR216H, ApR pCM235, waaLR216Q, ApR pCM235, waaLR216E, ApR g, b, and exo from l phage, araC-ParaB, ApR wbbLEcO16, SpR pCM234, waaL1-269:340-437FLAG (WaaLDEL5), ApR pCM235, waaL1-265:GFP, ApR pCM235, waaL1-419:GFP, ApR pCM235, waaLR288A, ApR pCM235, waaLY276A, ApR Source or reference Feldman et al. (2005) Laboratory Stock Neuhard and Thomassen (1976) This study Laboratory Stock Saldas et al. (2008) This study Lehrer et al. (2007) This study This study Datsenko and Wanner (2000) Valdivia et al. (1996) Valvano and Crosa (1989) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Datsenko and Wanner (2000) Feldman et al. (1999) This study This study This study This study This study

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

detected with anti-FLAG antibodies. Spheroplasts of cells expressing WaaLFLAG were isolated and incubated with trypsin for 2 or 4 h. Polypeptides of apparent masses of 19, 25 and 50 kDa were observed at 4 h digestion, which agree with the predicted molecular weights of cleavage products at EL5 and EL4, and the full-length undigested WaaL respectively (Fig. 4A, lane 2). Cleavage products containing the FLAG epitope were absent without trypsin or after 2 h digestion (Fig. 4A, lanes 3 and 4). As a control, spheroplasts were lysed with 0.1% Triton X-100, allowing trypsin to access the cytoplasmic and membrane regions of WaaL. Under these conditions, the amount of intact

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd, Molecular Microbiology, 70, 14241440

1428 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

W aa

A
kDa 119 91 51

FP G L

G FP

ut 3

38 29

Fig. 2. Topology of the WaaL protein based on C-terminal GFP fusions. Protein expression was examined by Western blot and uorescence microscopy. A. Western blot analysis using anti-GFP antiserum on total membranes isolated from E. coli K-12 DH5a cells expressing the WaaLGFP fusion 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 by uorescence microscopy at 1000 magnication as described in Experimental procedures. (B) Fluorescence microscopy, WaaLGFP (pMM4). (C) Fluorescence microscopy, GFPmut3 (pBADGFP).

WaaLFLAG was almost negligible and the 25 kDa cleavage product lost (Fig. 4A, lane 1). Also, we did not detect a ~3 kDa fragment corresponding to the predicted cleavage site in EL6 (data not shown). Therefore, trypsin cleavage of WaaL under native conditions is restricted to a limited amount of accessible cleavage sites in the periplasm yielding fragments containing the C-terminal FLAG epitope. The ~19 kDa C-terminal fragment, also found after digestion of Triton X-100-treated samples (Fig. 4A,

lane 1), suggests that cleavage occurs within the EL5. To conrm this, spheroplasts were prepared from E. coli cells expressing WaaLDEL5 (pMM1) and incubated with trypsin for 4 h. The Western blot revealed two bands of ~35 and ~42 kDa (Fig. 4B, lane 1). The ~35 kDa band likely corresponds to a C-terminal fragment from proteolysis occurring at any of the two cleavage sites within EL2, while the ~42 kDa band represents the undigested WaaLDEL5 protein. Cleavage products were absent without trypsin

A
D FL W AG aa L E L5 W aa L FL AG W aa L G FP pB A

B
L FL W aa AG L E L5 pB A D FL AG W aa

O antigen

206 119 91 51 38 29

core + 1 core
1 2 3 4
19

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 the O16 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 and silver 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 45C for 30 min. Lanes: M, Broad Range Prestained SDS-PAGE Standard (Bio-Rad); 1, WaaLFLAG (pCM234); 2, WaaLDEL5 (pMM1); 3, pBADFLAG. Asterisks indicate the migration of oligomeric forms of WaaL (see Results).
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Functional analysis of the WaaL large periplasmic loop 1429

Trypsin 4 Triton + 206 119 91

4 -

2 0 - -

B
Trypsin 4 0 4 0

Identication and functional analysis of conserved amino acids in EL5 To identify conserved residues in EL5 we used the Sequencing Alignment and Modelling system (SAM-T02), a suite of automated tools for modelling-, aligning- and discriminating-related protein sequences based on a linear hidden Markov model (Karplus et al., 2003). This analysis revealed that Arg-265, Asp-272, Gly-286, Arg288, Pro-303 and His-337 (the numbers denote the position of these residues in the E. coli K-12 WaaL) were the most conserved residues in proteins annotated as putative O antigen ligases and/or putative O antigen polymerases (Fig. 5A and data not shown). These conserved amino acids were individually replaced by alanine and the ability of each mutant protein to complement LPS O antigen surface expression in the DwaaL mutant CLM24(pMF19) was evaluated by silver staining and Western blotting with anti-O16- and anti-core OS-specic antibodies. LPS from the negative control, strain CLM24(pMF19, pBADFLAG), shows no O antigen detectable by silver staining or anti-core OS antibodies, while a small amount of O16-specic polymeric O antigen was detected with anti-O16 antibodies (Fig. 5B, arrow). This likely represents Und-PP-linked O16 antigen that cannot be ligated to lipid A-core OS. In contrast, LPS from the positive control, strain CLM24(pMF19, pCM235), forms polymeric O antigen and a band corresponding to core + 1, both of which are detectable by silver stain, anti-O16 and anti-core OS antibodies (Fig. 5B), indicating that WaaL function has been restored. Mutants R265A, D272A and G286A led to reduced O antigen surface expression, as evidenced by a reduced amount of polymeric O16 LPS and the lack of a detectable band corresponding to core + 1 (Fig. 5B). Polymeric O16 LPS was also detected using anti-core OS antibodies, suggesting that WaaL function is not completely impaired in these mutants. In contrast, the DwaaL strain carrying WaaL mutants H337A and R288A showed an even further reduction in polymeric O antigen, and more importantly, polymeric O antigen did not react with the core OS-specic antibody (Fig. 5B). This indicates that the O16-specic material produced by these mutants, which is weakly detected by silver stain (especially in the R288A mutant) but detectable with the anti-O16 serum, corresponds to Und-PP-linked polymeric O antigen not ligated to lipid A-core OS. The replacement P303A did not affect WaaL function, as the O antigen was identical to that produced by the parental strain (data not shown). The observed differences in LPS O antigen surface expression were not due to differences in the protein expression of the WaaL mutants (data not shown). Also, these different phenotypes were not due to loading artefacts since gel loading was normalized according to the bacterial

51 38 29 19 7
M 1 2 3 4
25 19

51 38

*
35 25 19

29
19 M 1 2 3 4

L5 E L W aa

Fig. 4. Periplasmic localization of EL5 by protease accessibility experiments in spheroplasts. Spheroplasts were treated with trypsin as indicated in each panel and cell lysates examined by Western blot with anti-FLAG M2 monoclonal antibodies. Asterisks in all panels indicate the migration of oligomeric forms of WaaL (see Results). 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 with trypsin. Digested samples were separated on a 16% Tris-Glycine SDS-PAGE. Arrows indicate the migration of the cleavage products. B. Spheroplasts of bacterial cells expressing WaaLFLAG were incubated with no trypsin (0) or with trypsin (4) for 4 h. Lanes: 1 and 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 the cleavage products.

(Fig. 4B, lanes 2 and 4). Together, the combined results of trypsin cleavage experiments of WaaL and WaaLDEL5, and the lack of GFP-mediated uorescence of WaaL1-R265-GFP indicate that EL5 is exposed to the periplasmic space. Larger bands of variable intensity reacting with the antiFLAG antibody were observed in all the Western blots (Figs 3B and 4A and B, asterisks). We interpreted these bands as corresponding to oligomeric forms of WaaL. This is not uncommon in membrane proteins, particularly in those proteins with high isoelectric points (Kashino, 2003). The mild denaturing conditions used for the preparation of the cell lysates, probably not sufficient to disperse protein oligomers, could account for the presence of larger bands, as we have also observed in previous studies with the integral membrane proteins WecA (Amer and Valvano, 2000; Lehrer et al., 2007), Wzx (Marolda et al., 2004) and WbaP (Saldas et al., 2008). These bands had a variation in mass that was proportional to the variation found in the respective monomeric proteins (Figs 3B and 4A and B, asterisks), suggesting that the presence of EL5 does not inuence the formation of the oligomeric forms of WaaL.

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd, Molecular Microbiology, 70, 14241440

W aa

LF

LA G

WaaL FLAG

1430 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

A
4 3 2 1

N K P I Q N R Y N E A L N D L N S Y T N A N S V T S L G A R L A MY E I G L N I F I K S P F S F R S
4 3 2 1

265

272

286 288

303

A E S R A E S MN L L V A E H N R L R G A L E F S N V H L H N E I I E A G S L K

337

B
AD pC M 2 R 35 26 5A D 27 2 R A 28 8 G A 28 H 6A 33 7 pB A AD pC M 2 R 35 26 5 D A 27 2 R A 28 8 G A 28 H 6A 33 7 pB A AD pC M 2 R 35 26 5 D A 27 2 R A 28 8 G A 28 H 6A 33 7A pB

O antigen

core + 1 core

Silver stain

Western anti-O16

Western anti-core OS

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 letter representing the amino acid is proportional to the level of conservation of this residue. The position of the most conserved residues is indicated below the letter (R265, D272, G286, R288, P303 and H337), and it was based on the automatic alignment by the server, using 55 sequences from putative WaaL homologues of sequenced strains of E. coli, S. exneri, S. boydii, S. dysenteriae, S. sonnei, Pectobacterium atrosepticum, Erwinia carotovora, Serratia marcescens, Klebsiella pneumoniae, Chromobacterium violaceum, Citrobacter koseri, Ralstonia metallidurans, R. picketti, R. solanacearum, Vibrio sp., Nitrosomonas europea, Nitrospira multiformis, Haemophilus inuenzae, Bordetella Petrii, Cupriavidus taiwanensis, Psychomonas sp., Comamonas testosteroni and Burkholderia phymatum. B. LPS prole 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 gel and stained with silver (Silver stain), or transferred to nitrocellulose membranes and reacted with polyclonal anti-O16-specic antibodies (Western anti-O16) or monoclonal anti-lipid A-core OS-specic antibodies (Western anti-core OS). 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. Arrow indicates polymeric O antigen that does not react with anti-core antibodies.

density at the starting point of the LPS preparations (note that the lipid A-core OS band in the silver-stained gel has the same intensity in all lanes; Fig. 5B). Previous work in our laboratory has shown that normalization by bacterial density is comparable to other methods including measurement of the concentration of 3-deoxy-D-mannooctulosonic acid (a conserved sugar component in the lipid A-core OS) or the amount of protein present in the whole-cell lysates before treatment with proteinase K (Vins et al., 2005). We also investigated the phenotypes mediated by the WaaL mutants in EL5 using the E. coli K-12 strain SCM3 carrying pJHCV32 (Table 1), which expresses O7 LPS. O16 and O7 are chemically distinct (Lvov et al., 1984; Stevenson et al., 1994). However, the same results as those observed with O16 LPS surface expression were

obtained in the presence of WaaL mutants R265A, D272A, G286A, R288A, P303A, H337A (data not shown), indicating that the observed phenotypes are independent of the composition and structure of the O antigen. Together, these experiments indicate that the conserved EL5 residues can be separated into three functional groups: residues that are required but not essential for full WaaL functionality (Arg265, Asp-272 and Gly-286), residues absolutely essential for function (Arg-288 and His-337) and residues with no functional effect on ligation (Pro-303). A three-dimensional structural model of EL5 predicts a putative catalytic centre To our knowledge, there is no structural information on WaaL proteins and also no crystallized structures with

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Functional analysis of the WaaL large periplasmic loop 1431

enough similarities to allow protein modelling by threading or other methods that rely on one or more evolutionary related protein structures as templates. Several secondary structure prediction algorithms predicted that EL5 is mostly a-helical, and most of the residues that are conserved in the alignments with WaaL homologues from other bacterial species are also present within predicted a-helices (data not shown). To gain further insight about a putative three-dimensional structural model of EL5, we employed de novo predictions. In this approach there is no strong dependence on database homology information, as the structural prediction relies on general principles governing protein structure and energetics. A de novo model of the EL5 from WaaLEcK12 was generated by the PROTINFO server of the University of Washington (Hung et al., 2005; 2007) using as input the sequence of residues Asn-259 to Lys-347 (Fig. 1). The proposed EL5 structural model consists of two pairs of almost perpendicular a-helices (Fig. 6). One a-helix from each pair (helices II and IV) contributes to form a structure containing a discrete region that is rich in positively charged amino acids exposed to the solvent. In particular, the model shows that most critical EL5 amino acids for ligase function, Arg-288 (contributed by a-helix II) and His-337 (contributed by a-helix IV), face each other (Fig. 6). The structural characteristics of this discrete region suggest it is a putative catalytic centre. To evaluate the predictive value of the model, several alanine replacements were constructed in residues situated in helices II and IV but away from Arg-288 and His-337 (Gly-295, Phe-299, Lys-301 and Gly-349) and also in the boundary between helices III and IV (His-324 and Asn-325) (Fig. 7A). Alanine replacements of these

residues resulted in mutant proteins that were correctly expressed in the membrane (data not shown) and did not affect WaaL function, as determined by the ability of each mutant protein to complement O16 LPS surface expression in CLM24(pMF19) (Fig. 7A, silver-stained gel). Additional alanine replacements were made in non-conserved amino acids located at positions surrounding the spatial location of Arg-288 and His-337. Tyr-276, Arg-328, His335 and Asn-338 were targeted in this region because of their chemical characteristics (positive charges and exposed OH groups). None of these replacements affected WaaL function, as compared with the parental protein (Fig. 7B, silver-stained gel). The conserved residues that when mutated resulted in reduced WaaL function (Fig. 5B) map to a-helices I (Arg265 and Asp-272) and II (Gly-286) (Fig. 6). A model of EL5 containing the G286A replacement showed that the distance between Arg-288 and His-337 in the mutant protein was greater than in the parental EL5 (5.3 in the parental EL5 and 13.6 in EL5G286A; Fig. 8). Similar alterations in spacing and angles of Arg-288 relative to His-337 were observed in models of EL5 with R265A and D272A replacements (data not shown). Together, the results suggest that these residues contribute to maintain the overall structure of the putative EL5 catalytic centre, in particular the spacing of the cross-over region of a-helices II and IV, dened by the proximity between the side-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 structural model, we also modelled the large periplasmic loop of

Front view
III IV
R288 P303 R265 H337

Back view
R288

IV III
H337

Top view
IV
R288 H337

III
P303

II

G286 G286

II
P303

II
G286

N
I

N
I
D272 R265

R265 D272 D272

C
PROTINFO).

Fig. 6. Structural model of EL5. De novo predicted tertiary structure of E. coli K-12 WaaL EL5 constructed using molecular dynamics (server
The model has been rotated to facilitate the visualization of the critical residues investigated in this work. Front, back and top view of 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 WaaL function; yellow, Pro-303 that if mutated to alanine does not affect WaaL function). N, N-terminus; C, C-terminus. The dotted line points to the C-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 plane suggesting continuity with the transmembrane segments.

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1432 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

5A G 34 9A

G 29

01

F2 9

32

32

A
O antigen

Front view
5A 9A 4A A
H324 N325 G295 F299 G295

Back view
N325 H324

K3

I
K301

F299

*
G349

*
G349

K301

core + 1 core

B
O antigen

8A

76

5A

8A

Y2

R328

32

33

33

R328

H335 H335

*
Y276

Y276

N338

core + 1 core

N338

Fig. 7. Functional analysis of non-conserved residues predicted to be near and far from the putative catalytic centre of EL5. The effect of these residues in WaaL function was examined by complementation of O16 LPS surface expression as determined by silver staining. 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. 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 of these 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 are located around the predicted catalytic centre (asterisk), but on the other side of helices II and IV, and were mutated to alanine.

WaaL proteins from R1 and R4 E. coli strains and compared them with the WaaLEcK12 model. Despite the low primary amino acid sequence similarity among these proteins, the models showed a similar structural arrangement and in all of them pairs of perpendicular a-helices were found (Fig. 9). Also, the conserved arginine residue corresponding to Arg-288 in WaaLEcK12 is part of a conserved GXR motif that according to the model is exposed to the solvent. This motif faces a conserved His residue that corresponds to His-337, within a conserved HXH motif. In the R1 and R4 ligase models the arginine and histidine residues were also contributed by helices II and IV respectively (Fig. 9). Highly similar tri-dimensional models obtained from dissimilar protein sequences strongly suggest a conserved tertiary structure in the large periplasmic loop of WaaL proteins. Unfortunately, modelling the predicted periplasmic loops of S. enterica serovar Typhimurium, E. coli R2 and E. coli R3 WaaL proteins

EL5

13.6 (G288A)

5.3 (EL5) EL5G288A

Fig. 8. Structural alignment of EL5 (green) and EL5 from the WaaLG288A mutant (pink) showing that the His-337 (blue) and Arg-288 (red) residues have altered spacing in the mutant protein.

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Functional analysis of the WaaL large periplasmic loop 1433

A
K12
II
R288 H337 R281

R1
N I
R288

R4
III
H337

I IV II C

IV IV III C N
H331

II C

III

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

II

III

IV

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. The amino acid sequences corresponding to the predicted large periplasmic loops were obtained and used for model generation. The a-helices I to IV are indicated, as well as the C-terminal (C) and N-terminal (N) ends of each protein. The models were rotated to facilitate visualization of the 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-helices I to IV, as indicated. Asterisks denote conserved residues in the three sequences. Dotted boxes indicate the GXR and HXH motifs in a-helices II and IV, respectively, containing the critical arginine and histidine residues.

afforded a large content of unstructured regions precluding comparisons among this group of ligases (data not shown). A positive charge in Arg-288 and in a conserved arginine of the predicted periplasmic loop 4 is required for WaaL activity Based on its location in the proposed catalytic centre and the strong phenotype of the R288A mutant, Arg-288 seems 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 the ligation reaction or is only required for structural purposes, we constructed replacements R288H, R288K, R288E and

R288Q. These mutant proteins led to different levels of LPS O antigen expression in silver-stained gels and in Western blots reacted with the anti-O16 antiserum (Fig. 10A). These differences could not be attributed to protein expression, since all the replacement mutant proteins had equal levels of expression and they were all inserted in the plasma membrane (data not shown). WaaLR288K was the only replacement that led to polymeric O antigen detectable with the anti-lipid A-core OS antiserum, suggesting that this mutant was able to restore O16 LPS surface expression (Fig. 10A). This result indicates that a positive charge in Arg-288 is required for WaaL activity. However, restoration of O16 LPS expression by WaaLR288K was not complete since as compared with parental WaaL, the amount of polymeric O antigen was highly reduced in bacteria containing the mutant protein.

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1434 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

A pC D M R 235 28 8 R Q 28 R 8K 28 R 8H 28 8E A pC D M R 235 28 R 8Q 28 8 R K 28 8 R H 28 8E pB A pC D M 23 5 R 28 8Q R 28 R 8K 28 8 R H 28 8E
Western anti-O16 Western anti-core OS

A
O antigen

core + 1 core
Silver stain

A pC D M R 235 21 6 R Q 21 R 6K 21 R 6H 21 6E

O antigen

core + 1 core
Silver stain Western anti-O16 Western anti-core OS

These differences could be attributed to the extended length of the aliphatic carbon chain of the lysine residue that could alter spacing relationships in the catalytic centre. The periplasmic loop 4 (EL4) of E. coli K-12 WaaL is predicted to consist of three amino acids (Fig. 1), one of which (Arg-216) is also highly conserved in all the ligases we have examined (data not shown). We investigated the function of this residue by constructing replacement mutants R216A, R216H, R216K, R216Q and R216E. Similar to the R288K replacement, WaaLR216K was the only derivative that remained functional as determined by the restoration of polymeric O antigen detectable by silver staining, an anti-O16 and anti-lipid A-core OS antibody (Fig. 10B and data not shown). This result demonstrates that a positive charge in Arg-216 is also critical for WaaL function.

Discussion
Using a combination of bioinformatics, C-terminal GFP fusions and protease accessibility experiments, we have derived a topological model of the E. coli K-12 WaaL, which consists of 12 predicted transmembrane helices and has the N- and C-termini oriented towards the cytoplasm. The experimental demonstration that the WaaL C-terminus is located in the cytoplasm agrees with the results of a genomic-scale topology analysis of inner membrane 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

A pC D M R 235 21 R 6Q 21 6 R K 21 6 R H 21 6E pB A pC D M 2 R 35 21 R 6Q 21 6 R K 21 6 R H 21 6E

pB

pB

Fig. 10. Functional role of positive charges in positions 288 and 216 in WaaL activity. LPS samples from CLM24/pMF19 cells expressing WaaL and WaaL mutants were run in a 14% Tricine-SDS gel and stained with silver (Silver stain), or transferred to nitrocellulose membranes and reacted with anti-O16-specic antibodies (Western anti-O16) or anti-lipid A-core OS-specic antibodies (Western anti-core OS). 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. Arrows indicate polymeric O antigen that does not react with anti-core antibodies. A. pBAD, WaaL (pCM235), and the WaaL mutants R288Q, R288K, R288H or R288E. B. pBAD, WaaL (pCM235), and the WaaL mutants R216Q, R216K, R216H or R216E.

pB

pB

assignment of EL4 and EL5 loops as exposed to the periplasm. EL5 is also analogous to the periplasmic loop IV of the V. cholerae WaaL (Schild et al., 2005) and the 49-amino-acid loop predicted in the WaaL protein from P. aeruginosa (Abeyrathne and Lam, 2007). A predicted large periplasmic loop similar to the EL5 of WaaLEcK12 is a common feature in other E. coli ligases (Heinrichs et al., 1998a) and also in a large number of putative ligases from a wide range of bacterial species, which were identied from genomic sequences in public databases (J.M. Prez and M.A. Valvano, unpublished). A relatively large periplasmic loop in WaaLEcK12 and other ligases strongly suggests that this loop has a functional role, which is consistent with the periplasmic location of the ligation reaction (Mulford and Osborn, 1983). Thus, it is not surprising that deletion of EL5 leads to the loss of WaaLEcK12 function. Comparative analyses with available sequences identied six residues in EL5 that are strongly conserved. From these, Arg-288 and His-337 showed the highest conservation. WaaL mutants with alanine replacements at any of these two positions showed the most dramatic effect in ligase function. In the majority of WaaL sequences from experimentally annotated ligases, and also in most WaaL proteins identied in genome sequences of many Gram-negative bacteria, residues corresponding to Arg-288 and His-337 are within conserved GXR and HXH motifs (J.M. Prez and M.A. Valvano, unpublished). Other ligases lacking the GXR and HXH motifs (such as the WaaL proteins from S. Typhimurium, S. Arizonae, S. Diarizonae, E. coli R2, E. coli R3 and Shigella exneri ) have SSYRY and

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Functional analysis of the WaaL large periplasmic loop 1435

SIQPHN motifs at equivalent positions, which also contain conserved arginine and histidine residues (underlined) respectively. These observations underscore the evolutionary conservation of the critical arginine and histidine residues in diverse WaaL proteins, suggesting these amino acids may play key roles in the ligation reaction. The loss or reduction in WaaL function in amino acid replacement mutants of the conserved residues in EL5 might be caused by a defective interaction of the enzyme with either Und-PP-linked substrates or the lipid A-core OS acceptor. It has been proposed that since a specic ligase can ligate distinct O antigens to the same lipid A-core molecules, the structure of the terminal core OS determines ligase specicity (Heinrichs et al., 1998a,c). Our results showing that the WaaL mutants caused the same LPS O antigen phenotypes with the O16 and O7 systems agree with this model and suggest it would be unlikely that the conserved residues of EL5 participate in recognition of core OS terminal sugars. Recently, it was reported that puried WaaL of P. aeruginosa possesses ATPase activity and ATP hydrolysis is required for the ligation reaction in vitro (Abeyrathne and Lam, 2007). Unfortunately, well-conserved motifs for ATP binding and/or hydrolysis are not apparent in the P. aeruginosa WaaL or in other WaaL proteins. Our efforts to mutate amino acids of regions in the E. coli K-12 WaaL showing a low level of similarity with Walker A and B motifs (Walker et al., 1982) and a palmate motif (Yamaguchi et al., 1993) did not result in any mutant protein with compromised WaaL function that could be attributable to ATP hydrolysis (Fig. S1). In any case, it would be unlikely that the conserved residues in EL5 are involved in ATP hydrolysis since ATP does not occur in the periplasm (Pugsley, 1993). Therefore, we propose that the conserved EL5 residues, which are common across WaaL proteins with different lipid A-core OS specicities, participate in the recognition of Und-PPlinked O antigen, and more specically the Und-PP moiety, as this is the only common part in the substrate molecules used by all WaaL proteins. Despite that at least one ligase protein has been successfully puried (Abeyrathne and Lam, 2007), structural data on these proteins are lacking. Furthermore, the low conservation in the amino acid sequence precludes structural predictions of the entire WaaL or subregions of the protein by comparative modelling and classical fold recognition methods. However, it is not unusual that periplasmic soluble regions between TM helices are structured (Lee et al., 2008; Parsons et al., 2008; Tocilj et al., 2008). Preliminary experiments to purify EL5 in quantities suited for structural analyses resulted in insoluble protein. Therefore, we reasoned that a molecular dynamics strategy (or de novo modelling) combined with mutagenesis could help establish a structural model

of the EL5 that could serve as a template to compare also with other WaaL proteins. De novo modelling is particularly strong for short peptides of less than 100 amino acids (Samudrala and Levitt, 2002), and can in principle be applied to recognize structural folds in solventexposed regions of membrane proteins. Our predicted tri-dimensional model of EL5 consists of two pairs of almost perpendicular a-helices. Comparisons with other proteins revealed low-level similarity between the predicted structure of EL5 and that of the catalytic domains in the ribose 5-phosphate isomerase (Zhang et al., 2003; Graille et al., 2005) and erythromycin polyketide synthase (Broadhurst et al., 2003). As predicted for EL5, these enzymes bind phosphorylated molecules as substrates or cofactors, respectively, and two pairs of a-helices arranged similarly to those in EL5 contribute to the catalytic domains. The highly conserved amino acids found in WaaL proteins, which led to a defective enzyme when replaced in the E. coli K-12 protein, are distributed within dened region of EL5. In particular, Arg-288 and His-337 are located facing each other. The orientation and the distance between Arg-288 and His-337 appear to be important for WaaL function. Indeed, according to our model, alanine replacements of neighbouring residues of the GXR motif affect the spatial orientation and distance of Arg-288 relative to His-337, while the global structure of the rest of the loop is not altered. This could explain the decreased ability of these WaaL mutants to complement O antigen synthesis. Also, a positive charge at position 288 is critical for WaaL function. From the various replacements at this position, only the R288K did not affect signicantly ligase function while other replacements resulted in a non-functional protein. Thus, we propose that the region containing the highly conserved residues in a-helices II and IV denes a putative catalytic reaction centre. Mutations reported in the literature in the conserved histidine residues of the periplasmic loops of WaaL from P. aeruginosa and V. cholerae (both corresponding to His-337) also affected ligase function in these systems (Schild et al., 2005; Abeyrathne and Lam, 2007), supporting even further the notion that this residue is critical for the activity of different ligases. As predicted from our model, additional alanine replacements of non-conserved EL5 residues both near and outside the putative catalytic region resulted in mutant WaaL forms that remained functional. Similarly, mutations made in non-conserved residues in the predicted periplasmic loop of the P. aeruginosa WaaL did not affect ligase function (Abeyrathne and Lam, 2007). WaaL proteins show signicant divergence in their primary amino acid sequence, even for members from the same species, but in contrast, hydrophobicity plot analyses show predictable secondary structural features

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1436 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

(Heinrichs et al., 1998a; Nesper et al., 2002). For instance, in E. coli there are ve chemically distinct types of lipid A-core OS (K-12, and R1 to R4) and strains with these types also have different WaaL proteins. We modelled the structure of the EL5 counterparts of WaaL proteins from E. coli R1 and R4 types. Remarkably, despite the divergence in the amino acid sequences of EL5-like loops the three models resulted in highly similar structures all consisting of four a-helices. Virtually all conserved amino acids were located in the same putative catalytic region and more importantly the critical residues at positions equivalent to Arg-288 and His-337 of WaaLEcK12, dened by the motifs GXR and HXH, are also placed facing each other. Therefore, our data support a conserved tertiary structure in the large periplasmic loop of the majority of WaaL proteins containing GXR and HXH motifs. Our study also shows that a highly conserved arginine residue (Arg-216 in WaaLEcK12) in the short periplasmic loop preceding the large loop also has signicant functional relevance. An arginine at an equivalent position in the V. cholerae WaaL was shown to be important for activity (Schild et al., 2005), and we demonstrate here that Arg-216 is required for WaaLEcK12 function. Moreover, similar to Arg-288, replacing Arg-216 with alanine, histidine, glutamine and aspartate resulted in non-functional proteins, while the R216K mutant was able to produce LPS O antigen. In summary, the results of this study lead us to propose that key EL5 and EL4 residues of WaaL may be part of a catalytic reaction centre and possibly involved in the binding of the phosphate groups of Und-PP. Indeed, histidine and lysine are critical active-site residues in other enzyme reactions resolving phosphodiester bonds, where these or similar residues act as proton donors or proton acceptors targeting the oxygen atom of one of the phosphate molecules. Similarly, the binding of ATP phosphate groups in the Walker A domains (R/KxxxxxGxxxL/VhhhD) is mediated by either lysine or arginine residues (Walker et al., 1982). Further experiments are in progress in our laboratory to explore whether or not Arg-288 and Arg-216 act in protonation/deprotonation on the phosphate bonds of the Und-PP-linked O antigens. The identication of a conserved structural model for the critical motifs found in WaaL and its corresponding periplasmic location provides a framework to dissect the WaaL reaction mechanism. Given the conservation of this structural motif, and the specic location of the uniquely conserved amino acids in the EL5 loop and in similar loops of many other WaaL proteins, we propose that these motifs are not involved in the specic recognition of the lipid A core acceptor molecules but rather participate in the chemical reaction or reactions required for the release of O antigen from the Und-PP lipid carrier.

Experimental procedures
Bacterial strains, plasmids and growth conditions
The plasmids and bacterial strains used in this study are listed in Table 1. Additional plasmids are listed in Table S1. Strain CLM24, an E. coli W3110 DwaaL, was used to assess protein expression of all constructs and for in vivo complementation studies. Bacteria were cultured at 37C in Luria Bertani (LB) medium supplemented with ampicillin (100 mg ml-1), tetracycline (20 mg ml-1) and 0.2% (w/v) arabinose, when appropriate. Transformation was performed by either the calcium chloride method or electroporation. All biochemical reagents were purchased from Sigma (St Louis, MO), unless indicated otherwise. Restriction endonucleases, T4 DNA ligase and associated buffers were purchased from Roche 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), carbonic anhydrase (38 kDa), soybean trypsin inhibitor (29 kDa) and lysozyme (19 kDa).

Construction of strains and plasmids

For complementation experiments using the LPS O7 system we constructed SCM3, a derivative of the E. coli strain S874 carrying a deletion of the waaL gene. The parental strain carries a large deletion that eliminates the E. coli K-12 O16 antigen biosynthesis cluster (Neuhard and Thomassen, 1976). This was performed as described by Datsenko and Wanner (2000). Primers corresponding to regions adjacent waaL (5-GCAGTTTTGGAAAAGTTATCATCATTATAAAGGT AAAACATGTGTAGGCTGGAGCTGCTTCG and 5-AGTGA GTTTTAACTCACTTCTTAAACTTGTTTATTCTTAACATATG AATATCCTCCTTAG) contained 20 additional nucleotides that annealed to the template DNA from plasmid pKD4 (in italics), which carries a kanamycin resistance gene anked by FRT (FLP recognition target) sites. Competent cells were prepared by growing S874 carrying pKD46 (Datsenko and Wanner, 2000) in LB containing 0.5% (w/v) arabinose, and the PCR products were introduced by electroporation. The plasmid pKD46 encodes the Red recombinase of the phage, which was placed under the control of the arabinoseinducible promoter PBAD. Kanamycin-resistant colonies were screened by PCR with primers annealing to regions outside of the mutated gene. Next, the antibiotic gene was excised by introducing the plasmid pCP20 (Datsenko and Wanner, 2000) encoding the FLP recombinase. Plasmids pKD46 and pCP20 are both thermosensitive for replication and were cured at 42C. The resulting strain was transformed with cosmid pJHCV32 carrying the genes encoding O7 LPS (Valvano and Crosa, 1989). Plasmid pCM234, encoding WaaL C-terminally fused to the Flag epitope (WaaLFLAG), was constructed by PCR amplication of a 1.27 kb fragment encoding waaL from E. coli strain W3110 with primers containing restriction sites (underlined) EcoRI (5-GACGGAATTCATGCTAACATCCT TTAAACTTC-3) and SmaI (5-CGTCCCGGGATTAATTGT ATTGTTACG-3). The PCR product was ligated (Rapid Ligation Kit, Roche Diagnostics) to pBADFLAG also digested with EcoRI and SmaI. Plasmid pCM235, encoding the WaaLFLAG

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Functional analysis of the WaaL large periplasmic loop 1437

C-terminally fused to a 5xHis tag, was constructed by amplication of a 732 bp fragment using pCM234 as a DNA template and vector primer (5-GATTAGCGGATCCTACC TGA-3) and XhoI primer (5-CGTCCTCGAGCTTGTCGTCG TCGTCGTC-3) restriction sites (underlined). The PCR product and the plasmid pBADHIS were digested with EcoRI and XhoI. To construct plasmid pMM1 encoding a deletion of the WaaL external loop 5 (WaaLDEL5) a 5.56 kb PCR product was amplied with pCM234 as DNA template and primers XhoI (5-CATGCTCGAGATTCTGTATTGGTTTATT3) and XhoI (5-GACTCTCGAGCATCTACATAATGAGATA3), digested with XhoI and self-ligated. Plasmid pBADGFP was created to construct C-terminal GFP fusions. A ~0.9 kb fragment encoding GFPmut3A was amplied from plasmid pFV25 using primers SmaI (5-GACTCCCGGGAGTAAAG GAGAAGAACTT-3) and PstI (5-CATTAAAGCTTGCATGC CTGCAGG-3). The PCR product and pBAD24 were digested with SmaI and PstI. Two plasmids carrying WaaLGFP fusion proteins were constructed: pMM3 (encoding WaaL1-265:GFP) and pMM4 (encoding WaaL1-419:GFP). In both cases the fragments were amplied from pCM234 DNA. The PCR products and pBADGFP were digested with EcoRI and SmaI. In all the cloning experiments the DNA fragments were ligated with T4 DNA ligase, and the ligation mix introduced into E. coli DH5a competent cells by transformation. Transformants were selected in media containing ampicillin and the appropriate plasmids were sequenced (York University Core Molecular Biology and DNA Sequencing Facility).

(Bio-Rad Protein Assay). Total membrane proteins were re-suspended, mixed with the appropriate amount of loading sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol and 0.1% bromophenol blue), and incubated at 45C for 30 min before loading. After separation on a SDS-PAGE, membrane proteins were transferred to nitrocellulose membranes for immunoblot analysis.

Western blots
Nitrocellulose membranes were blocked with 10% Western Blocking Solution (Roche diagnostics), followed by three 10 min washes with 50 ml of TBS pH 7.5 (50 mM Tris-HCL, 150 mM NaCl). Membranes were incubated for 2 h with the primary antibody: 7 mg ml-1 FLAG M2 monoclonal antibody (Sigma), 1:1000 dilution of O16 polyclonal antiserum, 1:5000 dilution of anti-core LPS monoclonal antibody (HyCult biotechnology b.v.), or 1:1000 dilution of anti-GFP polyclonal antibodies (Chemicon International), as appropriate. The reacting bands were detected by uorescence with an Odyssey infrared imaging system (Li-cor Biosciences) using IRDye800CW affinity-puried anti-rabbit IgG antibodies (Rockland, Pennsylvania) and Alexa Flour 680 anti-mouse IgG antibodies (Invitrogen).

Fluorescence microscopy
Five millilitres of fresh LB containing the appropriate antibiotic was inoculated to an OD of 0.15 from an overnight bacterial culture. Once bacteria reached mid-log phase, WaaL protein expression was induced with 0.02% (w/v) arabinose for 3 h. Bacteria were incubated on ice for 1 h to allow sufficient time for GFP to fold properly. Subsequently, 1012 ml of culture was transferred to a microscope slide coated with 0.8% agarose to slow down bacterial movement. Fluorescence and phase-contrast images were obtained using a Qimaging (Burnaby, British Columbia, Canada) cooled chargedcoupled device camera on an Axioscope 2 microscope (Carl Zeiss, Thornwood, NY). Live images were digitally processed with the Northern Eclipse version 6.0 imaging software (Empix Imaging, Mississauga, Ontario, Canada).

Site-directed mutagenesis
For site-directed mutagenesis of the EL5 of E. coli K-12 WaaL, oligonucleotide primers were designed to create the required amino acid change. The sequences of all primers used for mutagenesis are available from the authors upon request. The plasmid pCM235 was used as DNA template. PCR products amplied with Pfu polymerase were digested overnight with 1 U DpnI at 37C. The digested PCR products were introduced into DH5a competent cells by transformation, and transformants recovered by plating onto LB agar plates supplemented with ampicillin. Plasmids were recovered and the insert DNA was sequenced to conrm the introduction of the correct base-pair change encoding the amino acid substitution.

Isolation of spheroplasts
For spheroplasts isolation bacteria were freshly grown to an OD of 0.7. At this point protein expression was induced with 0.2% arabinose and incubated for 2 h at 37C. Cultures were placed on ice for 20 min, and 3 ml aliquots were centrifuged for 1 min at 16 100 g and the pellet re-suspended in 1 ml of an 18% sucrose solution in 0.001 mM Tris pH 8. Cell density was standardized to an OD600 of 2.0 in a 1 ml solution containing 18% (w/v) sucrose, 0.001 mM Tris pH 8, 10 mM EDTA and 100 mg ml-1 lysozyme. The suspension was incubated on ice for 30 min and the spheroplasts formation was monitored by light microscopy. Spheroplasts were collected by centrifugation (1 min at 16 100 g) and re-suspended in 70 ml of 18% sucrose in 0.001 mM Tris pH 8.0.

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-SDSPAGE and the gels stained with silver nitrate (Marolda et al., 2006). Total membranes were prepared from cells grown in LB 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, Roche Diagnostics) and they were lysed by sonic disruption for two 15 s pulses (Branson). Total membrane fractions were obtained by centrifugation of the lysates for 40 min at 40 000 g and the pellet re-suspended in the same buffer. Protein concentration was measured by the Bradford assay

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

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1438 J. M. Prez, M. A. McGarry, C. L. Marolda and M. A. Valvano

incubated from 2 to 4 h at 30C. Trypsin was inhibited with the addition of 0.1% Triuor Acetic acid (TFA) and 1 protein dye. Total lysate of spheroplasts was obtained with 0.1% Triton X-100. Digested products were identied by immunoblotting with 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 oligonucleotide primer design. CLUSTAL W was used for sequence alignments. BLAST searches were carried out using the tools provided by the national centre for biotechnology information. Hydrophobic patterns and topology predictions were performed using MEMSAT, PHD, TMHMM, TOPPRED and HMMTOP servers. For amino acids conservation studies and secondary structure predictions the HMM-based protein structure prediction server SAM TO2 was used (Karplus et al., 2003) with the input peptide sequence from spanning N259-K357 (Fig. 1). Molecular models of WaaL EL5 were constructed using molecular dynamics techniques provided by the PROTINFO server (http://protinfo.compbio.washington.edu) of the University of Washington. Structure visualization and electrochemical proles were performed using the program Pymol Molecular Graphics System (Delano Scientic LLC; http://pymol.sourceforge.net).

Acknowledgements
We thank M.S. Saldas, K. Patel, D.E. Heinrichs and C. Creuzenet for critical reading of the manuscript, and C. deLasa for constructing pBADGFP and WaaLGFP. This study was supported by a grant from the Canadian Institutes of Health Research. M.A.M. was supported in part by an Ontario Graduate Scholarship. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.

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