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LIPOPOLYSACCHARIDE ENDOTOXINS

Christian R. H. Raetz1 and Chris Whitfield2

1Department of Biochemistry, Duke University Medical Center, Durham, NorthCarolina 27710; e-mail: [email protected] of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada;e-mail: [email protected]

Key Words lipid A biosynthesis, Gram-negative bacteria, outer membranes,TLR4, LpxC, MsbA

f Abstract Bacterial lipopolysaccharides (LPS) typically consist of a hydropho-bic domain known as lipid A (or endotoxin), a nonrepeating “core” oligosaccharide,and a distal polysaccharide (or O-antigen). Recent genomic data have facilitatedstudy of LPS assembly in diverse Gram-negative bacteria, many of which are humanor plant pathogens, and have established the importance of lateral gene transfer ingenerating structural diversity of O-antigens. Many enzymes of lipid A biosynthesislike LpxC have been validated as targets for development of new antibiotics. Keygenes for lipid A biosynthesis have unexpectedly also been found in higher plants,indicating that eukaryotic lipid A-like molecules may exist. Most significant has beenthe identification of the plasma membrane protein TLR4 as the lipid A signalingreceptor of animal cells. TLR4 belongs to a family of innate immunity receptors thatpossess a large extracellular domain of leucine-rich repeats, a singletrans-membranesegment, and a smaller cytoplasmic signaling region that engages the adaptor proteinMyD88. The expanding knowledge of TLR4 specificity and its downstream signalingpathways should provide new opportunities for blocking inflammation associatedwith infection.

CONTENTS

ENDOTOXINS AS ACTIVATORS OF INNATE IMMUNITY . . . . . . . . . . . . 636LIPID A BIOSYNTHESIS IN ESCHERICHIA COLI AND SALMONELLATYPHIMURIUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641The Constitutive Lipid A (Endotoxin) Pathway. . . . . . . . . . . . . . . . . . . . . 641Regulated Pathways for the Covalent Modification of Lipid A. . . . . . . . . . . 643Origin of L-Ara4N-Modified Lipid A in Polymyxin Resistant Mutants. . . . . . 647

ROLE OF THE ABC TRANSPORTER MsbA IN LIPID A AND PHOSPHOLIPIDEXPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648

GENOMIC INSIGHTS INTO LIPID A BIOSYNTHESIS AND DIVERSITY . . . 651The Constitutive Lipid A Pathway As A Target For New Antibiotics. . . . . . . 651The Constitutive Pathway in Bacteria with Unusual Lipid A Structures. . . . . . 653Presence of Lipid A Biosynthesis Genes in Plants. . . . . . . . . . . . . . . . . . . 654

Annu. Rev. Biochem. 2002. 71:635–700DOI: 10.1146/annurev.biochem.71.110601.135414

Copyright © 2002 by Annual Reviews. All rights reservedFirst published as a Review in Advance on April 30, 2002

6350066-4154/02/0707-0635$14.00

STRUCTURE AND BIOSYNTHESIS OF CORE OLIGOSACCHARIDES . . . . . 655Structure of Core Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655Role of Core in Outer-Membrane Stability: The Deep-Rough Phenotype . . . . . 661Genetics and Biosynthesis of Core Oligosaccharide . . . . . . . . . . . . . . . . . . 662Assembly of the Inner Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665Assembly of the Outer Core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668Ligation of O Polysaccharide to Lipid A-Core Acceptor . . . . . . . . . . . . . . . 669Contributions of Core Biosynthesis to LPS Heterogeneity . . . . . . . . . . . . . . 670Phase Variation and the Biosynthesis of Lipooligosaccharide . . . . . . . . . . . . 671

STRUCTURE AND BIOSYNTHESIS OF O POLYSACCHARIDES. . . . . . . . . 672Structure of O Polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672Biosynthesis of O Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674Initiation Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675The Wzy-Dependent Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676The ABC-Transporter-Dependent Pathway. . . . . . . . . . . . . . . . . . . . . . . . 681The Synthase-Dependent Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684Seroconversion Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685

EXPORT OF LPS TO THE CELL SURFACE . . . . . . . . . . . . . . . . . . . . . . 688FUTURE DIRECTIONS FOR LPS RESEARCH . . . . . . . . . . . . . . . . . . . . . 689

ENDOTOXINS AS ACTIVATORS OF INNATEIMMUNITY

Lipid A (endotoxin), the hydrophobic anchor of lipopolysaccharide (LPS), is aglucosamine-based phospholipid that makes up the outer monolayer of the outermembranes of most Gram-negative bacteria (1–5). There are �106 lipid Aresidues and �107 glycerophospholipids in a single cell of Escherichia coli (6).The minimal LPS required for the growth of E. coli consists of the lipid A andKdo (3-deoxy-D-manno-oct-2-ulosonic acid) domains (Figures 1, 2) (1, 7, 8). Inwild-type strains, additional core and O-antigen sugars may be present (Figure 1)(5, 7, 9–11). Although generally not required for growth in the laboratory, thesehelp bacteria resist antibiotics, the complement system, and other environmentalstresses.

Many Gram-negative bacteria, including pathogens, synthesize lipid A speciesresembling the one found in E. coli (Figure 2) (1, 3, 4). Early ambiguitiesconcerning the structure of lipid A have generally been resolved [see (1, 3, 4)].Given their conserved architecture, most types of lipid A molecules are detectedat picomolar levels by an ancient receptor of the innate immune system presenton macrophages and endothelial animal cells (12, 13). The receptor, recentlyidentified as TLR4 (toll-like receptor 4) (14, 15), is a membrane-spanning proteinthat is distantly related to the IL1 receptor (12, 13).

In macrophages, lipid A activation of TLR4 triggers the biosynthesis ofdiverse mediators of inflammation, such as TNF-� and IL1-� (16, 17), andactivates the production of costimulatory molecules required for the adaptive

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637LIPOPOLYSACCHARIDE ENDOTOXINS

immune response (13). In mononuclear and endothelial cells, lipid A alsostimulates tissue factor production (18, 19). These events are desirable forclearing local infections, and they act in synergy. When overproduced systemi-cally in the setting of severe sepsis, however, the various mediators and clottingfactors can damage small blood vessels and precipitate Gram-negative septicshock, accompanied by disseminated intravascular coagulation and multipleorgan failure (20–23). Synthetic E. coli lipid A by itself causes a similarspectrum of effects when injected into animals (3, 24), supporting the proposedrole of lipid A in Gram-negative sepsis. The characteristic structural features ofE. coli lipid A (Figure 2), especially its two phosphate groups and its twoacyloxyacyl moieties, are needed to trigger the endotoxin response in humancells (3, 25–27).

Many of the initial events in the interaction of lipid A with animal cells havebeen elucidated in the past ten years (14, 15, 28, 29). A special lipid transferprotein in plasma delivers lipid A from bacteria (or bacterial membrane frag-ments) to CD14 on the surfaces of animal cells (Figure 3) (30, 31). Thesubsequent recognition of lipid A (or perhaps of the lipid A/CD14 complex) bythe receptor protein TLR4 represents the earliest known step in signal transduc-tion (Figure 3) (14, 15, 28, 29, 32). The interaction of lipid A with TLR4 likelyinvolves other proteins, including not only the phosphatidylinositol glycan-linkedsurface protein CD14 (33, 34) but also the soluble accessory protein MD2(Figure 3) (35). Despite very persuasive genetic evidence for the function ofTLR4 (14, 15), direct biochemical evidence for binding of lipid A and relatedmolecules to TLR4 is still lacking.

The first protein of the toll receptor family was discovered in the context ofinsect development but was later shown to play an additional role in protectinginsects against fungal infections (36–38). Subsequently, more than ten Toll-likereceptor protein homologues were discovered in the mouse and human genomes(12, 13, 39). Although the functions of the TLR proteins are incompletelyunderstood, TLR2 appears to be less specific than TLR4 in that it is activated bydiverse ligands, including bacterial lipoproteins and peptidoglycan fragments(12, 40). TLR6 may function together with TLR2 to recognize a subset ofbacterial membrane proteins and lipopeptides (41). TLR3 is activated by double-stranded RNA (42), and TLR5 is activated by bacterial flagellin (43). TLR9

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 2 Structure and biosynthesis of Kdo2-lipid A in E. coli K-12. The symbols indicatethe relevant structural genes encoding each of the enzymes (2, 48). A single enzymecatalyzes each reaction. The lipid A system may have evolved only once, as judged by theavailable genomes. In almost all cases, as illustrated by E. coli (367), the genes encodingthe enzymes of lipid A biosynthesis are present in single copy. At the protein level,orthologs of LpxA and LpxC are the most highly conserved among bacteria. The acyl chainincorporated by LpxA is highlighted in red. The LpxC inhibitor L-161,240 displaysantibiotic activity against E. coli that is comparable in potency to ampicillin (64, 65).


responds to bacterial DNA sequences containing the CpG motif (44). Assuggested for TLR4 in Figure 3, it may be that all Toll-like receptors function asdimers or perhaps as heterodimers, in analogy to the IL-1 receptor (45).

Although the three-dimensional structures of the leucine-rich extracellulardomains of the TLR receptors have not yet been determined, X-ray structures ofthe intracellular signaling domains of the TLR1 and TLR2 receptors haverecently been reported (46). These receptors show significant sequence homologyto the intracellular domain of the IL-1 receptor and closely resemble TLR4 intheir ability to engage the MyD88 adaptor protein (Figure 3).

LIPID A BIOSYNTHESIS IN ESCHERICHIA COLI ANDSALMONELLA TYPHIMURIUM

The Constitutive Lipid A (Endotoxin) Pathway

The enzymology and molecular genetics of the conserved steps of lipid Abiosynthesis are best characterized in E. coli (1, 2, 47, 48). The first reaction oflipid A biosynthesis is the acylation of the sugar nucleotide UDP-GlcNAc(Figure 2) (49). The E. coli UDP-GlcNAc acyltransferase (LpxA) is selective for�-hydroxymyristate, consistent with the composition of E. coli lipid A (49).UDP-GlcNAc acyltransferase has an absolute requirement for an acyl carrierprotein (ACP) thioester as its donor substrate (49, 50).

E. coli LpxA (a homotrimer) contains multiple contiguous hexad repeats (51),which are also found in certain other acyltransferases and acetyltransferases(52–54). The X-ray structure of LpxA shows that these hexads specify a novelprotein fold, consisting of a left-handed helix of short parallel �-sheets (51).Three hexads form one coil of the �-helix, and each LpxA monomer contains tensuch coils stacked on top of each other. The active site of E. coli LpxA, which

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 3 Detection of lipid A by the TLR4 innate immunity receptor of animal cells. Theparadigm is based mainly on studies of the human, mouse, and hamster immune cells andmacrophage lines (12–15, 27, 31, 368, 368a). We speculate that the TLR4 receptor may beoligomerized upon binding of lipid A. The TRL4-mediated inflammatory response isbeneficial in combating localized infections but may be detrimental in overwhelmingsystemic sepsis. Novel therapies of sepsis directed against the disseminated intravascularcoagulation component using recombinant activated protein C have recently shown efficacyin human trials (23). Combination therapy with potent TLR4 antagonists (369) remains tobe explored. In epithelial cells, the process of NF-�B activation may be somewhat different(details not shown) in that LPS apparently causes IKK activation inside the cell bytriggering the oligomerization of the plant disease resistance-like protein Nod1, followed byactivation of a distinct kinase known as RICK, which in turn activates IKK (369a).


is located between the coils of adjacent subunits, functions as an accuratehydrocarbon ruler that incorporates 14-carbon acyl chains two orders of magni-tude faster than 12- or 16-carbon acyl chains (55, 56). In Pseudomonas aerugi-nosa, the ruler is reset to measure 10 carbons (57). Single amino acidsubstitutions can convert P. aeruginosa LpxA to a 14-carbon-specific acyltrans-ferase or E. coli LpxA to a 10-carbon-specific enzyme (55). Chlamydia tracho-matis LpxA is unique in that it utilizes myristoyl-ACP instead ofhydroxymyristoyl-ACP, generating novel hybrid lipid A species when substi-tuted for E. coli LpxA in living cells (58). The structural basis for this selectivityis unknown.

The equilibrium constant (�0.01) for the acylation of UDP-GlcNAc isunfavorable (50). The biological implication is that the deacetylation of theproduct, UDP-3-O-(acyl)-GlcNAc (Figure 2), by the zinc metalloenzyme LpxCis the committed reaction of the pathway (59–63). LpxC is the product of aconserved, single-copy gene present in diverse Gram-negative bacteria, andit displays no sequence similarity to other deacetylases or amidases. LpxC isan excellent target for the design of novel, Gram-negative–specific antibiotics(64, 65).

Following deacetylation, a second �-hydroxymyristate moiety is incorporatedby LpxD to generate UDP-2,3-diacylglucosamine (Figure 2) (66). Again, onlyACP thioesters are substrates (66). The sequences of LpxA and LpxD aredistantly related because of the multiple, contiguous hexad repeats that arepresent in both proteins (66).

UDP-2,3-diacylglucosamine (Figure 2) is cleaved at its pyrophosphate bondby the highly selective pyrophosphatase LpxH to form 2,3-diacylglucosamine-1-phosphate (lipid X) (67). LpxH is missing in a few Gram-negative genomes,suggesting that additional enzymes of this kind may exist. A �, 1�-6 linkeddisaccharide is then generated by the condensation of another molecule ofUDP-2,3-diacylglucosamine with lipid X (Figure 2) (68, 69). The disaccharidesynthase is encoded by lpxB, which is cotranscribed with lpxA in E. coli (70–72)and many other organisms.

A specific kinase next phosphorylates the 4� position of the disaccharide toform lipid IVA (Figure 2) (73). The kinase structural gene (lpxK) (74, 75) islocated downstream in an operon with the msbA gene of E. coli. MsbA (seebelow) is an essential, inner-membrane ABC transporter (76) that functions in theinitial stages of lipid A (77, 78) and phospholipid (78, 79) export.

The kinase product, lipid IVA (Figure 2), is of great interest in its own rightbecause it possesses some of the properties of endotoxins (80). In mouse cells itis a potent endotoxin-like agonist, but in human cells it is an endotoxin antagonist(26). This unusual pharmacology of lipid IVA is determined by whether the targetcells express mouse or human TLR4 (Figure 3) (81, 82).

E. coli LPS contains two Kdo residues that are transferred to lipid IVA by abifunctional enzyme (Figure 2), encoded by waaA (also known as kdtA) (83). Therelated waaA gene of Haemophilus influenzae specifies an enzyme that adds one


Kdo moiety (84). It is not possible as yet to determine whether a Kdo transferaseis mono-, bi-, or even trifunctional (as in C. trachomatis) (85) by inspection ofits sequence. The labile sugar nucleotide, CMP-Kdo (1, 2), synthesized by KdsAand KdsB (not shown in Figure 2), serves as the Kdo donor.

The last steps of E. coli lipid A biosynthesis involve the addition of lauroyland myristoyl residues to the distal glucosamine unit (Figure 2), generatingacyloxyacyl moieties (86). The E. coli “ late” acyltransferases require the pres-ence of the Kdo disaccharide in their substrate for activity (86–88). Thissequence of events is not strictly conserved in P. aeruginosa (89), however, inwhich the ACP-dependent late acyltransferases add one secondary laurate chainto each glucosamine unit (Figure 4) in a symmetrical pattern. Like LpxA andLpxD, the late acyltransferases utilize acyl-ACPs exclusively as donors (86–88).The E. coli lauroyl and the myristoyl transferases are encoded by lpxL and lpxM,respectively (Figure 2), known as htrB and msbB prior to elucidation of theirfunctions (87–91), and display significant sequence similarity to each other, butnot to LpxA or LpxD. The lpxM(msbB) gene is not required for growth (91) inE. coli, but interestingly, lpxM mutants are greatly attenuated in their ability toactivate human macrophages (92). In Salmonella, lpxM mutants are much lesslethal than wild-type bacteria in animal shock models (93, 94), supporting acentral role for lipid A in sepsis physiology. S. typhimurium lpxM mutants, unliketheir E. coli counterparts, appear to harbor a second site suppressor(s) that allowsthem to grow (94a). Whatever the case may be, mutants with a reduced numbersecondary acyl chains on their lipid A moieties should be useful for thedevelopment of new vaccines (95).

Regulated Pathways for the Covalent Modification ofLipid A

E. coli contains one additional gene that is similar to lpxL, termed lpxP, whichis activated under conditions of cold shock (12°C) (96). LpxP inserts palmitoleatein place of laurate (not shown in Figure 2), perhaps reflecting the need to adjustouter-membrane fluidity at low temperatures (96). By deleting the lpxL, lpxM,and lpxP genes simultaneously, it is possible to obtain strains of E. coli with lipidA species that lack all secondary acyl chains (M. Vorachek-Warren & C.R.H.Raetz, in preparation). These triple mutants grow slowly on minimal medium butare not viable in nutrient broth. However, second site suppressors (M. Vorachek-Warren & C.R.H. Raetz, in preparation) or multicopy suppressors (see below)can be introduced that allow these E. coli mutants to grow almost normally underall conditions.

E. coli and Salmonella typhimurium contain additional enzymes for modifyinglipid A with phosphoethanolamine, 4-amino-4-deoxy-L-arabinose (L-Ara4N)and/or palmitate groups (Figure 4A). These enzymes are normally latent in E. coliK-12 but are expressed in the presence of metavanadate (97). Attachment of thephosphoethanolamine and L-Ara4N moieties is also induced by activation of thePmrA transcription factor following exposure to mildly acidic conditions, or by


mutation (98–102). The presence of the L-Ara4N group protects bacteria againstkilling by polymyxin and certain cationic antibacterial peptides (98, 99, 103)(Figure 4A). Polymyxin-resistance has provided a means to select mutants in thebiosynthesis and regulatory pathways.

The modification of lipid A with palmitate is under control of the PhoP/PhoQsystem (103, 104), which is activated by low concentrations of Mg2�, as wouldbe encountered inside phagolysomes (105, 106). S. typhimurium mutants unableto add palmitate to their lipid A are more sensitive than wild type to somecationic antimicrobial peptides (103, 103a). Interestingly, palmitate transfer tolipid A occurs in the outer membrane of S. typhimurium and E. coli (104) and iscatalyzed by the unusual palmitoyl transferase (PagP) (104) that uses glycero-phospholipids as its palmitoyl donor (107). The occurrence of PagP in eubacteriais limited to E. coli, Salmonella, Bordetella, Yersinia pestis, and Legionellapneumophila. Its absence in mutants of Legionella may interfere with pathogen-esis (108).


P. aeruginosa lipid A can also be modified with palmitate during conditionsof PhoP activation (Figure 4B) (109), but the palmitoyl group is added to adifferent site on lipid A than in E. coli (Figure 4A). P. aeruginosa does notcontain a PagP ortholog.

S. typhimurium contains two additional lipid A-modifying enzymes not foundin E. coli. The first of these LpxO hydroxylates the 3� secondary acyl chain in anO2-dependent manner (110) to generate S-2-hydroxymyristate (Figure 4A). Theother PagL selectively removes the �-hydroxymyristoyl residue at position 3(111) (Figure 4A). The functions of these modifications are unknown. Removalof the �-hydroxymyristoyl residue at position 3 might reduce the cytokine-inducing potential of bacterial cells.

The enzymes that add phosphoethanolamine to lipid A in polymyxin-resistantmutants (112) have not been characterized. However, E. coli cells grown with 5mM CaCl2 are induced to make a distinct phosphoethanolamine transferase (113)that modifies the outer Kdo residue (not shown). Phosphatidylethanolamine is thelikely phosphoethanolamine donor in all cases (102, 113), including the modi-fication of the heptose region (114), which occurs in a constitutive manner(Figure 1) (see below). The relevant genes have not been identified.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 4 Regulated modifications of lipid A in E. coli and S. typhimurium and unusuallipid A structures in other bacteria. Partial covalent modifications are indicated with thedashed bonds. Panel A. Modifications of E. coli and S. typhimurium lipid A under thecontrol of PmrA are blue, whereas modifications primarily under the control of PhoP arered. For a recent discussion of the structures of the modified lipid A species that can beisolated from various mutants or under different growth conditions see Zhou et al. (112).When present, the L-Ara4N (dark blue) moiety is located mainly at the 4� position, whereasthe phosphoethanolamine (light blue) is mostly at position 1. Certain lipid A species existin which the same substituents are attached at both sites, or in which their locations arereversed (112, 370). In cells grown with 1–10 mM Mg2� above pH 7.4, the modificationsare suppressed, and a pyrophosphate group (of unknown origin) is present at position 1 inabout one third of the lipid A molecules (112). Panel B. Lipid A modifications inPseudomonas under the control of PmrA are shown in blue, whereas modificationsprimarily under the control of PhoP are red (109). The portion of the lipid A moleculegenerated by the constitutive pathway is shown in black. Panel C. R. etli lipid A, whichlacks phosphate groups, includes a major species in which aminogluconate (magenta)replaces the proximal glucosamine. Aminogluconate is formed in the outer membrane froma lipid A species containing glucosamine (149, 150, 156). The 4� galacturonic acid moietyis green. Panel D. Like R. etli, Aquifex pyrophilus lipid A lacks phosphate moieties (126)but contains two galacturonic acid residues (green). The hydroxyacyl chains at positions 3and 3� differ from E. coli in that they are amide linked (magenta NH atoms).


Origin of L-Ara4N-Modified Lipid A in PolymyxinResistant Mutants

The proposed biosynthesis and mode of attachment of the L-Ara4N moiety tolipid A is shown in Figure 5. Our hypothesis for the existence of this scheme (97)was based on the discovery by Gunn et al. (99) of gene clusters in S. typhimuriumand E. coli required for the maintenance of polymyxin resistance. In accordancewith the system of Reeves et al. (9), the genes of the polymyxin resistance operonhave been renamed arn (Figure 5), given their recently demonstrated functions inthe biosynthesis and transfer of the L-Ara4N moiety (102, 115, 116). Thepathway starts with the conversion of UDP-glucose to UDP-glucuronic acid bythe well-characterized dehydrogenase, Ugd (Figure 5). Next, ArnA (previouslyOrf3 or PmrI) catalyzes the oxidative decarboxylation of UDP-glucuronic acid togenerate a novel UDP-4-keto-pyranose intermediate, which can be isolated inmilligram quantities (116). ArnB (previously Orf1 or PmrH) then catalyzes atransamination using glutamic acid as the amine donor to form UDP-L-Ara4N(116). Based upon its homology to dolichyl phosphate-mannose synthase ofyeast, it is believed that ArnC (PmrF) (99) transfers the L-Ara4N moiety toundecaprenyl phosphate (97), forming the novel compound undecaprenyl phos-phate-�-L-Ara4N. The existence of this important intermediate, which accumu-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 5 Pathway for L-Ara4N biosynthesis and mechanism of polymyxin resistance in E.coli and S. typhimurium. In accordance with the proposal of Reeves et al. (9), we haverenamed the genes of the polymyxin resistance operon “arn,” given their function in thebiosynthesis of the L-Ara4N moiety and its transfer to lipid A (97, 102, 115). The proposedpathway starts with the conversion of UDP-glucose to UDP-glucuronic acid by thewell-characterized dehydrogenase, Ugd. Next, ArnA (previously Orf3 or PmrI) (97, 102,115) catalyzes the oxidative decarboxylation of UDP-glucuronic acid to generate a novelUDP-4-keto-pyranose intermediate, which can be isolated in mg quantities using ArnA(116). In contrast to the proposal of Baker et al. (371), a separate enzyme to catalyze thedecarboxylation step is not necessary in our scheme (97, 116). ArnB (previously Orf1 orPmrH) (97, 99, 117) then catalyzes a further transamination to form UDP-L-Ara4N (116).Based upon its homology to dolichyl phosphate-mannose synthase of yeast, we propose thatArnC (PmrF) (97, 99, 117) transfers the L-Ara4N moiety to undecaprenyl phosphate,forming the novel compound undecaprenyl phosphate-�-L-Ara4N (102). After translocationto the outer surface of the inner membrane by unknown mechanisms, ArnT (previouslyOrf5, PmrK, or YfbI) (115) transfers the L-Ara4N unit to lipid A. Other genes of thepolymyxin operon (pmrJ, pmrL, and pmrM), as well as the adjacent pmrG gene (99, 117),cannot yet be assigned specific enzymatic or transport functions in our scheme. The ArnAprotein has a second catalytic domain (reaction not shown) that can transfer a formyl groupfrom N10-tetrahydrofolate to UDP-L-Ara4N (116), but the significance of this modificationis unclear. Addition of the L-Ara4N moiety to lipid A reduces the affinity of lipid A forpolymyxin and other cationic antimicrobial peptides.


lates in polymyxin resistant mutants, is firmly established (102). After itspresumed translocation to the outer surface of the inner membrane by unknownmechanisms (Figure 5), ArnT (previously Orf5, PmrK, or YfbI) transfers theL-Ara4N unit to lipid A (115). Robust in vitro assays for ArnT have recently beendeveloped (115). Other genes of the polymyxin resistance operon (pmrJ, pmrL,and pmrM), as well as the adjacent pmrG gene, cannot yet be assigned specificfunctions (99, 117).

Given that palmitate addition to lipid A occurs within the outer membrane(104) and L-Ara4N incorporation may take place within the periplasm (102, 115),it may be possible to utilize the presence or absence of these covalent modifi-cations as markers for the transport of nascent lipid A from its site of biosynthesison the inner surface of the inner membrane to the outer membrane (Figure 1).

ROLE OF THE ABC TRANSPORTER MsbA IN LIPID AAND PHOSPHOLIPID EXPORT

The outer membrane is an asymmetric bilayer with phospholipids on its innersurface, and lipid A, the hydrophobic anchor of LPS on the outside (Figure 1).The constitutive enzymes that make phospholipids and lipid A (Figure 2) in E.coli are located in the cytoplasm or on the inner surface of the inner membrane(1, 118). How E. coli lipids cross the inner membrane and are transported to theouter membrane is not understood at the molecular level.

A clue to LPS transport has recently emerged from studies of E. colilpxL(htrB) mutants and their suppression by multiple copies of the msbA gene(77–79, 87, 119). LpxL is the lauroyl transferase that functions late in E. colilipid A biosynthesis (Figure 2) (87, 119). Lipopolysaccharides bearing tetra-acylated lipid A species accumulate in the inner membranes of lpxL mutants at42°C, and unless second site suppressors are introduced, cell growth is inhibited(78). MsbA is an essential ABC transporter (Figures 6, 7), closely related toeukaryotic MDR proteins (76). MsbA overexpression represents one way inwhich to restore growth of lpxL mutants at 42°C without restoring laurateincorporation (78), resulting in the export of lipopolysaccharides with tetra-acylated lipid A anchors to the outer membrane (78). E. coli msbA knockouts arelethal. Their biochemical analysis is complicated by the long times (4–8 h)needed to dilute out pre-existing MsbA supplied in trans from a temperature-sensitive plasmid (78) and by the fact that the lpxK gene (Figure 2), which isimmediately downstream in an operon with MsbA in E. coli, is also essential forcell growth (75).

To gain a clearer understanding of the function of MsbA, a new temperature-sensitive mutant of E. coli (WD2) was recently isolated in which there is a singleA270T substitution in the fifth trans-membrane region of MsbA, and the effectof this mutation has been studied in detail (79) (Figure 6). This MsbA variant israpidly inactivated at 44°C. As shown by 32Pi and 14C-acetate labeling, export of


Figure 6 Structure of the E. coli MsbA dimer at 4.5 Å resolution. This backbone tracingwas made from protein data bank file 1JSQ (120). Trans-membrane helices 1–6 are coloredpurple, blue, yellow, green, red, and orange, respectively. The intracellular domain (ICD)is brown, and the nucleotide-binding domain (NBD) is cyan (120). A schematic model oflipid A is shown in panel A for size comparison. The location of the A270 residue, whichis changed to threonine in the temperature sensitive lipid transport mutant WD2 (79), isshown as a red sphere. The putative chamber for binding lipids on the inner surface of theinner membrane is lined with basic residues (120) (not shown). Exactly how MsbA mightfacilitate lipid flip-flop in the cytoplasmic membrane and/or export to the outer membranein an ATP-dependent manner is unknown (120, 371a)


all major lipids (i.e., both LPS and phospholipids) to the outer membrane isinhibited by �90% in WD2 after a 30-min shift to 44°C. Transport of newlysynthesized proteins is not impaired under the same conditions. Electron micros-copy reveals inner-membrane lamellae and invaginations in WD2 at 44°C,consistent with an increased surface area secondary to the complete block of lipidexport (79).

His-tagged versions of MsbA have been constructed. The protein was over-expressed, solubilized with nonionic detergents, purified, and reconstituted.Purified MsbA catalyzes lipid stimulated ATP hydrolysis in vitro. Kdo2-lipid Ais an especially potent activator (W. Doerrler & C.R.H. Raetz, unpublished),suggesting that this molecule is transported. Cell-free assays to evaluate MsbAcatalyzed lipid flip-flop and/or intervesicular lipid transport remain to be estab-lished.

Chang & Roth recently purified and crystallized E. coli MsbA using a similarpreparation method, and they solved its structure at a resolution of 4.5 Å (Figure6) (120). This remarkable achievement should provide insights into the functionsof lipid pumps, including the mammalian MDR proteins, which are about twiceas long as MsbA but are internally duplicated (120, 121). Based on the crystalstructure (Figure 6), MsbA is a homodimer (120). It contains six trans-membranehelices (consistent with hydropathy analysis) (79). A large cavity between thesubunits could accommodate lipid A and/or phospholipid molecules (Figure 6A).This space is lined with basic residues (120). Exactly how MsbA would mediatelipid flip-flop by binding and hydrolyzing ATP in the nucleotide binding domains(Figure 6) is unclear. It is speculated that the nucleotide binding domains maycome together following lipid entry into the putative transport chamber presentin the trans-membrane region (Figure 6), forcing the lipid to move to theperiplasmic side of the inner membrane (as suggested in Figure 7).

MsbA may indeed function as a flippase within the inner membrane, but inprinciple, it could also be a pump that ejects lipid A and phospholipids from theouter surface of the inner membrane during the transit of these substances to theouter membrane (Figure 7). The location of the A270T mutation at the criticalMsbA dimer interface on the periplasmic surface of the inner membrane (Figure6) is compatible with the lipid export defect observed in mutant WD2 (79). Weanticipate that additional, as yet unidentified, protein components are needed toshuttle lipids across the periplasm and to assemble them properly in the outermembrane (Figure 7, steps 2, 3). Although the details will surely differ, the TolChemolysin secretion system represents an interesting paradigm with respect to theinvolvement of multiple membrane proteins in the export process, including anABC transporter with similarity to MsbA (76, 122).

Although many proteins in the MDR family appear to catalyze lipid flip-flopin vitro, mouse knockout mutants lacking the three major MDR proteins show nogeneralized defects in lipid trafficking, and the mice are viable (121). MDR2knockouts do display a specific phospholipid transport deficiency in that theycannot pump phosphatidylcholine into their bile (123). Several distinct ABC


transporters have recently been shown to be involved in reverse cholesterol flowand bile acid secretion (124). We believe that the many additional MDR-likeproteins, present in all eucaryotes, may function in catalyzing lipid traffickingwhen the three known MDR proteins are inactivated. It may be necessary toisolate mutants defective in more than just the three major MDR proteins toobtain generalized lipid transport defects in animal cells, resembling those seenin WD2.

Other bacterial ABC transporters play key roles in exporting some types ofO-antigens and bacterial capsules (see below). As with MsbA, robust in vitrosystems to study the biochemistry of these transporters are needed.

GENOMIC INSIGHTS INTO LIPID A BIOSYNTHESISAND DIVERSITY

The Constitutive Lipid A Pathway As A Target ForNew Antibiotics

The genomic sequences of about 100 Gram-negative bacteria are either completeor nearing completion. In almost every case, each of the constitutive enzymes oflipid A biosynthesis (Figure 2) is encoded by a single-copy gene. LpxH (67) is

Figure 7 Model for MsbA-mediated lipid export in E. coli. Following the MsbA-mediated transport at the inner membrane, additional proteins are likely to beinvolved in steps 2 and 3, but these have not yet been identified (79).


present in only �70% of the available genomes, suggesting that additionalisoenzymes catalyzing UDP-2,3,-diacylglucosamine hydrolysis (Figure 2) mayexist. A few of the genomes also lack homologues of lpxL and/or lpxM (125),though at least one acyloxyacyl moiety is invariably present in all lipid As whenanalyzed chemically (126). LpxA and LpxC are the most highly conserved of theconstitutive enzymes (Figure 2). The enzymes that catalyze regulated or otherspecial modifications of lipid A (Figures 4, 5) are more variable and are restrictedin their distribution.

Lipid A is required for growth of E. coli and most other Gram-negativebacteria (6, 64) studied so far; furthermore, it is essential for the maintenance ofan effective outer membrane barrier (127). The outer-membrane protein FhuAinteracts in a highly specific manner with LPS (128, 129), and lipid A may berequired for the proper folding of some porins (130). Inhibitors of lipid Abiosynthesis are themselves good antibiotics against E. coli, and sensitizebacteria to other antibiotics that normally do not penetrate the outer membrane(64). Onishi et al. have described L-161,240 (64), a specific LpxC inhibitor witha Ki of about 50 nM (Figure 2, inset) that is bactericidal against E. coli atconcentrations comparable to ampicillin (64, 65). This compound also killsstrains of Enterobacter and Klebsiella, but not Pseudomonas (64, 65). Resistancein the latter case is due to the fact that L-161,240 and related compounds are poorinhibitors of P. aeruginosa LpxC. Novel inhibitors that are active against diverseLpxCs have recently been designed but have not yet been optimized (65). AnX-ray structure of LpxC would be very helpful in this regard. LpxC is a solublezinc containing metalloenzyme (62, 63), implying that far more potent inhibitorsmight yet be found, as demonstrated in earlier studies with angiotensin convert-ing enzyme (131) and carboxypeptidase A (132).

In contrast to E. coli and most other Gram-negative human pathogens, strainsof Neisseria meningitidis that possess a polysialic acid capsule can grow slowlywithout lipid A when their lpxA gene is inactivated (133). Such cells no longeractivate TLR4 but do activate TLR2 at �100-fold higher doses (134). In suchmutants, the polysialicacid capsule becomes essential for growth (134a) andappears to substitute for lipid A. The hydrophobic anchor of the polysialic acidcapsule of N. menigitidis is not fully characterized (135). These remarkableobservations are important in that they may provide new insights into thefunctions of lipid A in Gram-negative bacteria.

A few types of diverse bacteria that possess outer membranes inherently lackthe lipid A system, as defined by the absence of the lpx genes (Figure 2). Theseinclude Sphingomonas paucimobilis, which appears to contain glycosphingolip-ids in place of lipid A (136); Thermotoga maritima, the genome of whichcontains about 25% archaebacterial sequences (137); Deinococcus radiodurans(138, 139); and the spirochetes Treponema pallidum and Borrelia burgdorferi(140, 141). The genomic information for B. burgdorferi is consistent with thefailure to detect lipid A in this organism by chemical methods (142). Whetherother lipids substitute for lipid A in these organisms (as in Sphingomonas) is


unknown. Some bacteria that lack the lpx genes, such as Deinococcus radio-durans, do contain “alien gene” clusters encoding putative glycosyl transferases,resembling O-polysaccharide biosynthesis modules (143, 144). Such O-polysac-charide-like polymers, which could function as receptors for bacteriophages (seebelow), might be grafted onto a novel lipid anchor(s) in these systems. Theunderlying biochemistry has not yet been explored. Unlike O-polysaccharidemodules, the lpx genes themselves (Figure 2) do not qualify as alien (143),suggesting that they have not undergone extensive lateral transfer.

T. pallidum and B. burgdorferi have relatively small genomes (140, 141) andare restricted in their ability to grow outside of their hosts. On the other hand,Leptospira interrogans is a spirochete with a much larger genome that can growon a defined medium (145, 146). It contains the genes encoding the constitutivelipid A pathway (Figure 2). The final product may be modified in a uniquemanner, however, given that L. interrogans lipid A appears to activate TLR2instead of TLR4 (146).

The Constitutive Pathway in Bacteria with Unusual Lipid AStructures

As shown in Figure 4, significant variations in lipid A structure occur in someGram-negative bacteria. These changes (relative to E. coli and S. typhimurium)include differences in acyl chain length and distribution as in P. aeruginosa (Figure4B) (109, 147), the absence of phosphate moieties in organisms like Rhizobium etli(148–150) and Aquifex pyrophilus (126) (Figure 4C, D), the presence of galacturonicacid residues (Figure 4C, D), and subtle modifications of one or both glucosamineunits (Figure 4C, D) (126, 148–150). Despite these differences, the key enzymes ofthe constitutive lipid A pathway (Figure 2) remain operative (65, 125, 151). Theobserved structural variations are generally explained by the existence of additionalenzymes (152–157) that are not present in E. coli.

In P. aeruginosa, the altered substrate specificities of LpxA and LpxD account forthe shorter R-3-hydroxyacyl chains (Figure 4B) (158). The LpxL ortholog thatincorporates the secondary laurate on the proximal glucosamine unit is Kdo inde-pendent (89). The secondary laurate chain on the distal unit is incorporated by a moretypical Kdo-dependent LpxL ortholog (N. Que & C.R.H. Raetz, in preparation). TwoLpxO orthologs (110) are present in P. aeruginosa, possibly accounting for the twoalternative sites at which 2-hydroxylaurate may be generated (159) (Figure 4B, redX) in an O2-dependent manner (160). In contrast to the secondary laurate residues,the secondary palmitoyl chain of P. aeruginosa lipid A (Figure 4B, red) isnot incorporated by an ACP-dependent mechanism (S. Trent & C. R. H. Raetz,unpublished), and it is absent unless the PhoP system is activated (109). However, aPagP ortholog is not present in P. aeruginosa. Similarly, although there is noapparent PagL ortholog in Pseudomonas, much of the 3-position is deacylated inmature LPS species isolated from cells (109, 147, 161) (Figure 4B). A 3-O-deacylaseactivity is present in cell extracts (S. Trent & C. R. H. Raetz, unpublished). Incontrast to the situation with PagP and PagL, the orthologs responsible for L-Ara4N


modifications (Figure 4, dark blue) of P. aeruginosa appear to be very similar tothose of E. coli and S. typhimurium (Figure 5).

R. etli lipid A consists of several molecular species (149, 150), the mostunusual of which are shown in Figure 4C. The absence of phosphate residues isexplained by late-functioning phosphatases (152, 153), perhaps located on theouter surface of the inner membrane (157). The oxidation of the dephosphory-lated proximal residue to generate the aminogluconate moiety (Figure 4C,magenta) occurs in the outer membrane (156). The long secondary (C28) acylchain at position 2� is incorporated by a distant ortholog of LpxL (S. Basu &C.R.H. Raetz, in preparation), which requires a specialized acyl carrier protein(ACP-XL) (155). The origins of the pendant 3-hydroxybutryate moiety and the4� galacturonic acid residue (Figure 4C, green) are unknown.

Like R. etli, A. pyrophilus lipid A lacks phosphate residues and containsgalacturonic acid moieties (Figure 4D, green) (126). In addition, the glucosaminedisaccharide is replaced with two 2,3-diamino-2,3-dideoxy-D-glucose residues(Figure 4D, magenta NH atoms) (126). The latter are also found in many otherGram-negative bacteria (162), e.g., Thiobacillus ferrooxidans. In the latterorganism, the 2,3-diamino-2,3-dideoxy-D-glucose residue arises by NAD-depen-dent oxidation of UDP-GlcNAc at the GlcN 3-position, followed by a transam-ination with glutamic acid as the amine donor (163). The resulting “UDP-3-amino-3-deoxy-GlcNAc” is then utilized preferentially by Thiobacillus LpxA, inanalogy to what occurs with UDP-GlcNAc in the E. coli pathway (Figure 2) (C.Sweet & C.R.H. Raetz, unpublished).

The biological significance of these and other lipid A structural variationsawait evaluation by genetic methods. The unusual R. etli lipid A might beadvantageous during nodulation and symbiosis with plants, whereas A. pyrophi-lus lipid A might confer thermotolerance. Elucidation of the relevant enzymologyis a necessary prelude to genetic studies.

Presence of Lipid A Biosynthesis Genes in Plants

Recent versions of protein, DNA, and EST databases indicate that Arabidopsisthaliana and other plants contain significant homologues of E. coli genesencoding enzymes of lipid A biosynthesis. These genomic observations implythat plants synthesize lipid A-like substance(s). Lipid A or closely relatedmolecules may be minor constituents of plant membranes that were overlookedin earlier biochemical studies. The lipid A pathway may have appeared in plantsfollowing symbiosis with cyanobacteria. Nuclear genes encoding apparentorthologs of LpxA, LpxC, LpxB, LpxD, LpxK, KdtA, KdsA, and KdsB arepresent in the genomic DNA database of A. thaliana. These genes have intronsand display plant codon usage. ESTs encoding key portions of LpxA, LpxC,LpxD, LpxB, LpxK, and KdtA have also been recovered from many other plants,including cotton, pine, rice, soybean, pepper, and corn. Known active-siteresidues of LpxA (56) and LpxC (63) are conserved.


The plant homologues of the E. coli lpx gene products display 30% to 45%sequence identity over the predicted lengths of each of the proteins. Given theenzymatic studies of diverse bacterial Lpx proteins (58, 65, 85), this degree ofsequence identity between plants and E. coli is indicative of a closely relatedenzymatic function. The A. thaliana lpxA gene can in fact complement atemperature-sensitive mutant of E. coli with a point mutation in its own lpxAgene (D. Liu & C.R.H. Raetz, unpublished) (6).

The subcellular localization of the plant lpx gene products is uncertain. LpxAcontains an N-terminal leader sequence in A. thaliana suggestive of targeting tochloroplasts or mitochondria. Interestingly, full-length lpxA and lpxC genes areactually found next to each other in the chloroplast genome of the red algaCyanidium caldarium (164). Perhaps, the first few steps of lipid A biosynthesisoccur in chloroplasts, where ACP and fatty acids are abundantly available (165).

Given these considerations, it is reasonable to hypothesize that plants have thecapacity to make a lipid A-like molecule, minimally consisting of Kdo-lipid IVA

(Figure 2). However, extensive additional modifications to this scaffold mightoccur, as is observed in some other Gram-negative bacteria (Figure 4C, D) (126,149, 150). Orthologs of the lipid A genes are not present in the human, mouse,worm, fly, or yeast databases.

What functions might plant lipid A serve? One scenario is that it is a structuralcomponent of chloroplast outer membranes. Alternatively, lipid A could function asa signaling molecule in plants, given that homologues of LPS binding protein and ofthe extracellular domains of the Toll-like receptors are found in A. thaliana. OneTLR homologue of Arabidopsis has been identified as a brassinosteroid receptor(166), which is required for normal growth. The brassinosteroid receptor is homol-ogous to other plant gene products needed for disease resistance (166).

Characterization of the lpxA gene knockouts in A. thaliana (D. Liu & C. R. H.Raetz, unpublished), in conjunction with a systematic search for lipid A-likesubstances, should provide incisive information concerning the function of theLpx orthologs in plants. Given that lipid A-like molecules are potent adjuvantsin humans, the possible existence of such molecules in plants may prove to be ofconsiderable medical/toxicological interest with regard to the development ofallergies or respiratory diseases induced by plant dust (167).

STRUCTURE AND BIOSYNTHESIS OFCORE OLIGOSACCHARIDES

Structure of Core Oligosaccharides

As with lipid A, much of our understanding of the structure and biosynthesis ofthe core oligosaccharide is founded on work in E. coli and Salmonella, but


detailed structures are now available from a variety of organisms with differentlifestyles, and the scope of biosynthesis data is rapidly expanding. The structuresof core oligosaccharides are described in detail elsewhere [reviewed in (168)],and only a general overview is provided here. In bacteria that produce smoothLPS (S-LPS), the core oligosaccharides are conceptually divided into tworegions: inner core (lipid A proximal) and outer core. The outer core regionprovides an attachment site for O polysaccharide (O antigen). Mucosal pathogensoften lack O polysaccharides. Instead they produce lipooligosaccharides (LOSs)that contain a recognizable inner core from which extend one or more mono- oroligosaccharide branches (equivalent to the outer core). These extensions deter-mine serological specificity [for detailed coverage of the structure and functionof LOS, see (169–171, a)].

Within a genus or family, the structure of the inner core tends to be wellconserved, and the fact that the core oligosaccharides from distantly relatedbacteria share structural features in the inner core is a reflection of the importanceof the core in outer-membrane integrity. The inner core typically containsresidues of Kdo and L-glycero-D-mannoheptose (L,D-Hep) (Figure 8, inset). TheKdo residue is the only component found in all known cores, although in somecases a derivative, D-glycero-D-talo-oct-ulosonic acid (Ko), is also present (5).Some bacteria contain D-glycero-D-mannoheptose (D,D-Hep) alone or in com-bination with the more prevalent L,D-Hep (see Figure 9), whereas others, likeRhizobium, lack heptose entirely (154, 172, 173). The base structure of inner coreis often decorated with nonstoichiometric additions of other sugars and withphosphate (P), pyrophosphorylethanolamine (2-aminoethanol; PPEtN), or phos-phorylcholine (PCho) residues. The varying extent of these modifications con-tributes to the heterogeneity of LPS molecules extracted from a culture. In manycases, only the structure of the predominant carbohydrate backbone is known fora given core oligosaccharide, and the extent of phosphorylation and nonstoichio-metric additions are unknown. This is a reflection of the difficulties encounteredin structural analyses.

The outer core shows more structural diversity, as might be expected for aregion with more exposure to the selective pressures of host responses, bacte-riophages, and environmental stresses. However, the extent of structural variationin core oligosaccharides within a given species, or even a genus, is still limited.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 8 Structures of the known lipopolysaccharide core oligosaccharides from E. coliand Salmonella. The outer cores are shown, together with one heptose residue of the innercore. The ligation sites for O polysaccharides (O-PS) are indicated where known [reviewedin (201)]. The inset shows the conserved base structure of the inner core, and type-specificnonstoichiometric additions to the inner core are identified by dotted lines. Residues andlinkages that are conserved in each of the core oligosaccharides are shown in red. Detailsof the structures are described elsewhere (168, 236, 372). Unless otherwise noted, alllinkages are in the �-anomeric configuration.


For example, in E. coli there are five known core types (R1, R2, R3, R4, andK-12) (Figure 8). All are found among commensal isolates (174), whereas the R1type predominates among strains that cause extraintestinal infections (175, 176)and the R3 type is found in most verotoxigenic isolates such as O157:H7 (174,177). These cores differ in the nonstoichiometric inner-core substituents found ina minor fraction of isolated LPS molecules, but the most substantial changes areevident in the outer core (Figure 8). The two known cores from Salmonella arequite similar to those of E. coli (Figure 8), and genetic data indicate that thestructure found in serovar Typhimurium is common to isolates from routinehuman infections (S. enterica subspecies I), whereas the structure found inArizonae IIIA predominates in other subspecies (N. Kaniuk & C. Whitfield,unpublished). In K. pneumoniae only one major core structure has been discov-ered (Figure 9), and this is distributed among different serotypes [(178, 179) andreferences therein]. However, there is some variation in the extent of nonstoi-chiometric substitutions among isolates, and there may be some additional corediversity based on reactivity with a core-specific monoclonal antibody (180). TheK. pneumoniae core with its outer core �-Kdo provides an example of a structurewith lability to both mild acid and alkaline deacylation, two treatments com-monly used to generate carbohydrate backbones for structural analysis. As aresult, some features of the outer-core structure were not identified in initialanalyses (178). The limited structural variation in the core oligosaccharide withina genus is in striking contrast to the hypervariable O polysaccharides and hasstimulated interest in the possibility of targeting the core oligosaccharides togenerate immunotherapeutic antibodies (181–185). Similarly, the enzymesresponsible for the conserved structural features in inner cores provide potentialtargets for novel therapeutics.

In the LOS from mucosal pathogens, the inner core is also conserved withina species. However, differential expression of the attached oligosaccharides leadsto phase variation and multiple glycoforms and immunotypes in the LOS (170,171). The LOS of N. meningitidis provides a good example, where the varyingcomposition of the �-chain and differential substitution of the side-branchheptose (�- and �-chains), and sialylation all generate heterogeneity (Figure 10).In several cases LOS contain structures that mimic host-cell antigens.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 9 Structures of the core oligosaccharides from K. pneumoniae O1 and P.aeruginosa O5. The �-galacturonic acid residues on the K. pneumoniae core are nonstoi-chiometric, and details of the structures are described elsewhere [(178, 179, 232, 373) andreferences therein]. The boxed regions of the core structures are those that differ betweenS-LPS and R-LPS, and the details are discussed in the text.


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Role of Core in Outer-Membrane Stability:The Deep-Rough Phenotype

The deep-rough phenotype is displayed by E. coli and Salmonella mutants thatlack the heptose region of the inner core. The phenotype is actually a series ofcharacteristics that collectively reflect changes in both structure and compositionof the outer membrane leading to its instability [reviewed in (7, 186)]. Thesecharacteristics include changes in surface hydrophilicity, resulting in hypersen-sitivity to hydrophobic dyes, detergents, hydrophobic antibiotics, fatty acids,phenols, and polycyclic hydrocarbons. Deep-rough mutants also typically releasesignificant amounts of periplasmic enzymes into the culture media unless highconcentrations of Mg2� are included in the medium. The outer membrane ofsome deep-rough mutants has also been reported to have a decreased proteincontent with a concomitant increase in phospholipid. Finally, deep-roughmutants of E. coli activate expression of colanic acid exopolysaccharide, loseexpression of pili and flagella (187), and secrete a form of hemolysin withreduced hemolytic activity (188, 189). These factors can also influence biofilmformation (190).

Early studies on the deep-rough phenotype used Salmonella mutants withmultiple LPS defects leading to truncated molecules with reduced phosphoryla-tion. Phosphoryl groups were implicated in the formation of a stable membranethrough the provision of sites that would enable adjacent LPS molecules to becross-linked by divalent cations, and it is well established that treatment of cellswith EDTA has a detrimental effect on outer membrane integrity and viability(186). In fact, for some time it was thought that mutants lacking heptose-Presidues were not viable unless the core was truncated. Discovery and biochem-ical characterization of the LPS kinase WaaP (see below) allowed construction ofdefined mutants lacking core phosphorylation but retaining a complete carbohy-drate backbone and, where relevant, the O polysaccharide (191, 192). In thelaboratory, waaP mutants show the same growth rate as the parent. Interestingly,waaP mutants in E. coli show some characteristics of the deep-rough phenotype(e.g., increased sensitivity to hydrophobic compounds) but do not show alteredouter-membrane protein profiles (193). Mutations that eliminate synthesis of theouter core result in increased susceptibility to some hydrophobic compounds dueto an indirect effect on core phosphorylation (193). The absence of corephosphorylation attenuates the virulence of S. enterica serovar Typhimurium,and this is thought to be due in part to increased sensitivity to polycationicpeptides. In this respect, pmrAB-regulated modifications of the lipid A-core thataccompany polymyxin resistance cannot overcome the defects resulting from lostphosphorylation (191). Collectively, these results have confirmed the criticalroles played by phosphoryl groups in the LPS of E. coli and Salmonella and havehelped clarify their association with the deep-rough phenotype.

While E. coli and Salmonella can accommodate LPS lacking core phosphor-ylation, P. aeruginosa mutants with defects in waaP or inner-core heptose


assembly are not viable (194). The P. aeruginosa core has a multiply phosphor-ylated inner-core heptose region (195) (Figure 9), and this organism is highlysusceptible to lysis in EDTA (196). Thus phosphorylation is more critical insome bacteria than in others, and more examples are needed before we candetermine whether the ability of E. coli and Salmonella to tolerate phosphory-lation defects is broadly representative. Other bacteria apparently accommodatethe requirement for a negatively charged inner core in different ways. In K.pneumoniae, there is no phosphorylation of the heptose region, and equivalentnegative charges may be provided by uronic acids (Figure 9). The same is trueof R. etli (173). Structural heterogeneity in the LPS extracted from K. pneu-moniae results from structural variants lacking one or both of the terminal�-galacturonic acid residues (178, 179). Notably, K. pneumoniae typically shedssignificant amounts of an extracellular toxic complex comprising capsular poly-saccharide, LPS, and proteins, and this complex is implicated in the damage tolung tissues that characterizes the pneumonia caused by this organism (197, 198).It will be interesting to determine whether these �-galacturonic acid substitutionsare essential for viability or if their elimination results in a deep-rough pheno-type. The Bordetella pertussis core also lacks heptose-P but does have Kdo-P anduronic acids (199). Deep-rough mutants have also been constructed in B.pertussis, parapertussis, and bronchiseptica by mutating the gene (waaF) whoseproduct adds the second heptose residue in the core backbone (200). Thesemutants lack the remainder of the core that, in this organism, is attached to theremaining heptose residue. The mutants are viable, but properties reflecting thestability of the outer membrane have not been reported.

Genetics and Biosynthesis of Core Oligosaccharide

The completed lipid A-Kdo2 serves as the acceptor on which the core oligosac-charide and LOS chains are assembled via sequential glycosyl transfer fromnucleotide sugar precursors. Endogenous in vitro glycosyltransferase activity canbe detected in membrane fractions, and many of the enzymes are predicted to beperipheral proteins. It is assumed that rapid and efficient core oligosaccharidesynthesis reflects a coordinated complex of membrane-associated glycosyltrans-ferases, although the existence of such a complex has not been tested experi-mentally. The complex would act at the cytoplasmic face of the inner membrane,where acceptor and nucleotide sugars are available. Given the diversity of knownstructures, biosynthetic functions have not been assigned in all cases.

The chromosomal waa region (formerly rfa) contains the major core-oligo-saccharide assembly operons, and E. coli K-12 provided the first waa regionsequenced in its entirety. Although some waa-encoded functions have beendefined biochemically in E. coli K-12, assignment of others relies heavily on LPSstructure and LPS-specific phage-receptor data for Salmonella mutants and onheterologous complementation of those mutations. Comparative sequence anal-ysis of the waa regions from type strains representing the known core oligosac-charide types of E. coli and S. enterica serovar Typhimurium has been reported,


and the insertions, deletions, and rearrangements that result in the different corestructures have been discussed [reviewed in (201)]. Homologs of core biosyn-thetic genes are now being identified in a variety of nonenteric bacteria, andadvances in whole-genome sequencing will result in a rapid expansion of data inthis area. Since many gene assignments currently rely heavily on E. coli andSalmonella prototypes, a consideration of the limitations of the functionalassignments in these prototypes is critical. Arguably, the best-characterizedsystem involves the E. coli R1 core where all of the genes in major corebiosynthesis operon have been mutated and the structure of the resulting LPS hasbeen determined (Figure 11). Some of the R1 enzymes have also been studied ata biochemical level. From this prototype, and using data for conserved enzymesin other E. coli and Salmonella core systems, a comprehensive picture isemerging.

In E. coli, Salmonella, and K. pneumoniae (201, 202), the identification ofcore biosynthesis genes is aided by the fact that they are clustered on thechromosome. These loci generally encode all of the activities required forouter-core assembly as well as the transferases needed for inner-core synthesis.In E. coli and Salmonella, the waa locus consists of three operons (Figure 11)mapping between cysE and pyrE on the chromosome. The operons are defined bythe first gene in each transcriptional unit: gmhD-, waaQ-, and waaA. ThegmhD-waaFC genes are required for biosynthesis and transfer of L,D-heptose (seebelow). Transcription of the gmhD operon in E. coli K-12 is regulated by aheat-shock promoter, perhaps indicating a requirement for the heptose domain ofLPS, at least in E. coli K-12, for growth at elevated temperatures (2, 7). The longcentral waaQ operon contains genes necessary for the biosynthesis of the outercore and for modification and/or decoration of core. In E. coli isolates with theR1 and R4 cores, this operon also contains the “ ligase” structural gene (waaL),whose product is required to link O polysaccharide to the completed core. ThewaaQ operon is preceded by a JUMPStart (Just Upstream of Many Polysaccha-ride-associated gene Starts) sequence that includes the conserved 8-bp regionknown as ops (operon polarity suppressor) that, together with RfaH (a NusGhomolog), is required for operon polarity suppression [reviewed in (203, 204)].As expected, Salmonella and E. coli K-12 mutants deficient in RfaH producetruncated LPS molecules [reviewed in (7)] due to premature termination withinthe operon and reduced amounts of some outer-core synthesis enzymes. ThewaaA transcript contains the structural gene (waaA, formerly kdtA) for thebifunctional Kdo transferase (83) and a “non-LPS” gene encoding phospho-pantetheine adenylyltransferase (coaD, formerly kdtB) (205).

Examination of annotated genomes from more distantly related organismsindicates that some have clusters of subsets of core genes; examples includeCampylobacter jejuni, Vibrio cholerae, and P. aeruginosa. Determination of therange of functions encoded by these loci and identification of “missing” genes arelimited by the number of ORFs with no currently known function. In mucosalpathogens including Haemophilus influenzae and N. meningitidis, there is no


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significant clustering of inner-core biosynthesis genes, but many of the genesencoding glycosyltransferases that synthesize serologically important oligosac-charide extensions (e.g., LgtA-E, Figure 10) tend to be found in the same locus.

Assembly of the Inner Core

The Kdo transferase, WaaA (formerly KdtA) is discussed above because,although Kdo is a core component, its assembly in the nascent LPS moleculeprecedes completion of lipid A in E. coli.

In the early 1980s, ADP-D,D-heptose and ADP-L,D-heptose were isolated fromS. enterica serovar Minnesota and Shigella sonnei (206, 207), identifying theprecursor for heptose residues. Using synthetic substrates and in vitro reactions,it has been confirmed that the configuration of L,D-Hep residues in the core isestablished by the specificity of the heptosyltransferases, WaaC and WaaF. Theseenzymes exhibit preference for ADP-L,D-heptose as their substrate, with ADP-D,D-heptose being used at low efficiency (208, 209). Interestingly, ADP-D-mannose can also serve as a surrogate substrate for WaaC (210). WaaC andWaaF homologs have been identified in a variety of bacteria, and in most cases,convincing assignments have been made by complementation of the correspond-ing mutations in Salmonella and E. coli.

Despite identification of the transferases and the L,D-heptose precursor, thepathway for ADP-L,D-heptose synthesis has only recently been resolved (Figure12). The initial step was predicted in a pathway for NDP-heptose synthesis firstproposed in 1971 (211) and involves conversion of sedheptulose-7-phosphate toD,D-heptose-7-phosphate by the isomerase, GmhA. This activity has been con-firmed in both E. coli and H. influenzae, (212, 213). The first clue that theoriginally proposed pathway was not strictly correct came from work on RfaE,an enzyme acting downstream of GmhA that is essential for formation ofheptose-containing LPS (214). The sequence of this enzyme predicted twodomains, one with similarity to ribokinases and the other resembling nucleoti-dyltransferases. The bifunctional nature of this enzyme, now renamed HldE, wassubsequently confirmed (215). These activities correlated with data for theparallel pathway for formation of GDP-D,D-heptose, a precursor for the glyco-sylation of S-layer proteins in Aneurinibacillus thermoaerophilus (216). Theauthors have proposed a modified pathway (Figure 12) involving a kinasereaction to generate a D,D-heptose-1,7- bisphosphate intermediate and invokingan additional phosphatase step to generate D,D-heptose-1-phosphate. The previ-ously undetected E. coli phosphatase (GmhB) was found by database searchesusing the corresponding A. thermoaerophilus sequence, and its activity wasconfirmed biochemically and by analysis of a gmhB chromosomal mutation(215). Sequential activities of GmhA-HldE-GmhB-HldE lead to the formation ofADP-D-glycero-D-manno-heptose. The terminal epimerase enzyme (HldD, for-merly RfaD) converts this compound to the preferred heptosyltransferase sub-strate ADP-L-glycero-D-manno-heptose. HldD been characterized at thebiochemical (217) and structural (218) levels.


Elucidation of the A. thermoaerophilus pathway was aided by the fact that thegenes encoding the enzymes for GDP-D,D-heptose synthesis are clustered on thechromosome. The same seems to be the case for the ADP-heptose genes in C.jejuni and Helicobacter pylori, whereas the homologs from E. coli, P. aerugi-nosa, and H. influenzae are spread throughout the chromosome. Interestingly,while most of the homologs are well conserved in Gram-negative bacteria, N.meningitidis has the two domains of HldE in separate polypeptides (214). V.cholerae contains the isomerase and phosphatase but lacks obvious candidatesfor the kinase and nucleotidytransferase activities (216), despite the presence ofL,D-heptose residues in its core oligosaccharide [reviewed in (168)] and

Figure 12 Biosynthesis pathway for ADP-L-glycero-D-manno-heptose in E. coli.The figure is adapted from recent studies that necessitate revision of the previouslyproposed pathway (215).


homologs of known heptosyltransferases in the genome sequence. Nevertheless,the widespread distribution of these enzymes in important pathogens, the require-ment for heptose-containing LPS in the formation of a stable outer membraneand the now-established synthetic pathway offer new avenues for the develop-ment of therapeutic compounds.

The modification of the core region of E. coli and Salmonella LPS requires theaction of three enzymes, WaaP (an LPS kinase), WaaY (an enzyme required fora second phosphorylation), and WaaQ (a transferase that adds the side-branchheptose) (Figure 11). The WaaP enzyme is the most important of these activitiessince the modifications must proceed in the strict order WaaPQY (192), and asindicated, mutants lacking WaaP activity exhibit the deep-rough phenotype andare avirulent. The WaaP protein has been purified, and its kinase action estab-lished in vitro using radiolabeled ATP and an acceptor consisting of isolatedwaaP mutant LPS (219). Although WaaP shares little primary sequence homol-ogy with eukaryotic kinases, PSI-BLAST searches did identify some conserva-tion in catalytic residues inferred from functional and structural studies ofeukaryotic kinases. Site-directed mutagenesis confirmed that these residues arealso essential for catalysis in WaaP. The WaaP enzyme from P. aeruginosashares extensive similarity with the homologs from E. coli and Salmonella, andthey are functionally interchangeable (194). However, the P. aeruginosa WaaPenzyme also appears to exhibit autokinase activity (194a). Additional putativekinases have been identified in P. aeruginosa, and these are presumably respon-sible for the remaining phosphates in the core oligosaccharide of that organism.What remains to be determined is the timing of these modifications duringsynthesis and the nature of the preferred acceptor. The ordered events inWaaPQY activity suggest complex acceptor requirements for the modifyingenzymes. Mutant LPSs lacking the outer core (waaG in Figure 11) are phos-phorylated inefficiently by WaaP (�40% of wild-type levels) (193). Conversely,the loss of phosphorylation results in a decreased efficiency of core extension,leading to more truncated cores than seen in the wild type, so there may becomplex protein:protein interactions required for optimal core assembly. TheWaaP kinase is not homologous to the Kdo kinase (KdkA) that phosphorylatesthe 4� position of the single Kdo residue of H. influenzae (220, 221).

The modifications of inner core that involve addition of glycose substituentsto Kdo residues (Figure 8) have received less attention. The WaaZ enzyme isfound in S. enterica and in E. coli isolates with the R2 and K-12 core types andis responsible for the ��2,4 Kdo transferase activity that adds the third Kdoresidue found only in the inner cores of these bacteria (E. Fridrich, B. Lindner,O. Holst & C. Whitfield, unpublished results). Putative glycosyltransferases havebeen identified for the other inner-core modification reactions, but functional dataare currently limited [reviewed in (201)]. In laboratory-grown cultures, the LPScontains only minor amounts of molecules with these modifications, making theiridentification particularly difficult.


Assembly of the Outer Core

All of the E. coli and Salmonella cores have a glucose residue as the first sugarin the outer core. Biochemical data, LPS structure, and heterologous genecomplementation experiments identify WaaG as the UDP-glucose:(heptosyl)LPS �1,3-glucosyltransferase in E. coli K-12 (222) and Salmonella [reviewed in(201)]. As expected, the homologs of WaaG from each of the core types arehighly conserved. The subsequent two transferases form �1,3- and �1,2-link-ages, respectively, and depending on the resulting structure (Figure 8), theenzymes transfer glucose or galactose to an acceptor of either glucose orgalactose. The corresponding transferases are all members of family 8 in theglycosyltransferases classification system (223) (http://afmb.cnrs-mrs.fr/�pedro/CAZY/) and share several highly conserved motifs (201), making assignments oftheir functions difficult in the absence of either an LPS structure from a definedmutant or biochemical evidence with a purified protein. These enzymes areretaining glycosyltransferases, catalyzing formation of an �-linkage in theproduct from an �-linked donor. The mechanism of action of these transferasesis not fully understood despite the interesting insight provided by the recentstructural elucidation of a family 8 representative, LgtC (224). LgtC is an�1,4-galactosyltransferase involved in LOS synthesis in N. meningitidis, and theprotein was crystalized in the presence of both a sugar nucleotide donor andacceptor analogues, allowing mechanistic predictions to be tested. The prevailingmodel for the activity of retaining transferases was inferred from detailed studiesconcerning glycosylhydrolases and was proposed to involve a double displace-ment reaction involving a transient covalent intermediate between the donorsugar and the enzyme. However, an appropriately placed catalytic nucleophilecould not be identified on the face of LgtC in proximity to the donor. The authorsproposed an alternative model in which departing and attacking groups exist onthe same side of the sugar ring. Their simultaneous action would result in aSNi-like internal return transition state.

In general, the enzymes catalyzing conserved linkages in E. coli and S.enterica cores are closely related, and the genes occupy similar positions in theloci. One exception is the glycosyltransferases responsible for the terminal�-1,2-linked glucose in E. coli R3 and S. enterica serovar Arizonae IIIA (Figure8). The enzymes responsible in these strains share no significant similarity andare located at different positions within the corresponding waaQ operons (N.Kaniuk & C. Whitfield, unpublished results).

Within E. coli and Salmonella, the R1 and R4 core types are the only onesinvolving a �-glycosyltransferase. These inverting enzymes use the �-linkeddonor to generate a �-linked product. They are also identified by sequencesimilarity and two-dimensional features evident in Hydrophobic Cluster Analysis(HCA) (225), and the structures for several examples have been reported[reviewed in (226, 227)]. These enzymes require (at least) two catalytic carboxyl-ates, where one serves to coordinate a divalent cation associated with the donor


sugar nucleotide and another serves as a base to activate the donor. The R1 andR4 systems add a different �-linked sugar at the same site in an otherwiseidentical outer core (Figures 8 and 11), and the enzymes (WaaV and WaaX,respectively) were identified by interchanging them and determining the resultingLPS structure (228). These enzymes belong to different glycosyltransferasefamilies (WaaV, family 2; WaaX, family 25).

Ligation of O Polysaccharide to Lipid A-Core Acceptor

Completion of the S-LPS molecule involves the addition of O polysaccharideto the nascent lipid A-core, and the ligation reaction occurs at the periplasmicface of the cytoplasmic membrane [reviewed in (11, 201)]. Lipid A-core isdelivered to the periplasmic face by MsbA (see above), and the threepathways for O-polysaccharide synthesis take different approaches to delivercompleted O polysaccharide to this location; these are described below. Theligation reaction mechanism has not been determined, and the waaL geneproduct is currently the only enzyme presumed to be required for the process.However, in vitro assays with well-defined donor and acceptor substrateshave not been reported.

WaaL primary sequences offer no obvious clues as to function. Collec-tively, they share only low levels of similarity in their primary sequences, butthey are all predicted to be integral membrane proteins with 8 or moremembrane-spanning domains. The resulting hydropathy profiles are virtuallyidentical with hydrophobic domains of similar length and distribution.Although ligase enzymes form glycosidic linkages, the donor is an undeca-prenol pyrophosphate-linked oligo- or polysaccharide, and consequently,ligases share no similarity with any of the families of glycosyltransferasesthat utilize sugar nucleotide donors. The ligase enzyme presumably functionsas part of a complex that involves highly specific interactions with thelipid-linked O-polysaccharide intermediates and lipid A-core acceptor. Theligase enzyme from E. coli K-12 can link to its lipid A-core O polysaccha-rides with diverse structures, arising from any of the known biosynthesispathways. The lack of discrimination for donor structures suggests that theligase enzyme may recognize the undecaprenol pyrophosphate carrier ratherthan the polymer attached to it. The variations in ligase sequences may thenreflect specificity for the lipid A-core acceptor structure, or required protein:protein interactions in the biosynthesis complex, or both. Acceptor specificityhas been demonstrated and involves not just the core residue providing theattachment site, but also other proximal residues in the core (229 –231). Theligation sites in the E. coli cores are variable (Figure 8). There are nosignificant correlations between the structure of the linkage site and WaaLsequence. And heterologous complementation of waaL defects is only pos-sible if the complementing gene product acts on a closely related corestructure.


Contributions of Core Biosynthesis to LPS Heterogeneity

LPS molecules extracted from a given isolate show variation in the extent of corecompletion. The extent of capping of lipid A-cores with O polysaccharide (i.e.,the amount of rough vs. smooth LPS) can also vary in a strain-dependent manner.It has been suggested that the heterogeneity of LPS is affected by modificationsin the inner-core Kdo region (Figure 8) [reviewed in (7)]. Analysis of SDS-PAGE profiles of LPS from mutants of E. coli K-12 led to speculation that WaaS(a putative 1,5 rhamnosyltransferase), WaaZ (the putative distal �-2,4 Kdotransferase), and WaaQ play an integral role in the production of two forms ofLPS. In this scenario, the classical LPS form provides an acceptor for Opolysaccharide, whereas the “LOS form” (distinct from R-LPS) is destined toterminate without O-polysaccharide addition. There are no current biochemicaldata that directly support this interesting hypothesis. The WaaZ enzyme isconserved in E. coli isolates with K-12 and R2 cores and in S. enterica, and allthree are reported to contain some LPS molecules bearing a Kdo3 moiety (Figure8). Conversely, the R1 core type lacks the waaZ gene and has no third Kdo in itsnatural structure. When plasmid-encoded WaaZ activity is introduced into abackground with R1 core LPS, the Kdo3 structure becomes evident and most coreoligosaccharides truncate prematurely in the outer core (E. Fridrich, B. Lindner,O. Holst, and C. Whitfield, unpublished results). Although these results suggesta role for the inner-core modification in control of core extension, the data couldalso be explained by WaaZ overexpression disrupting important protein:proteininteractions and stoichiometry in a core biosynthesis enzyme complex. Furtherresearch is required to resolve these alternatives.

Differences in outer core can also dictate the efficiency of ligation of Oantigen to the core, in turn influencing LPS heterogeneity. In P. aeruginosa thereare significant differences in the structures of core with, and without, attached Opolysaccharides (232) (Figure 9). R-LPS cores contain a �-D-Glc-(132)-�-L-Rha-moiety that is absent in the smooth LPS. Conversely, only the S-LPScontains an essential �-(133)-linked L-Rha residue that provides the ligation site(233). Recent studies have also identified core modifications involving quorumsensing-dependent expression of migA, encoding a putative glycosyltransferase(234), but the resulting structures have not been determined. Overexpression ofMigA results in loss of molecules containing single-repeat units of O polysac-charide, with no apparent effect on longer chain molecules, and the gene is highlyexpressed in the cystic fibrosis lung, suggesting a biologically significant role. InK. pneumoniae, R-LPS molecules contain a heptan structure and lack the �-Kdoresidue in the outer core that serves as the ligation site (178, 179, 235, 235a; E.Vinogradov, E. Fridrich, L. L. MacLean, M. B. Perry, O. Petersen, C. Whitfield,manuscript in preparation). In S. enterica serovar Arizonae IIIA, smooth LPSmolecules have been reported to lack the terminal �-(132)-linked Glc residue(Figure 8), and the possibility that this residue may be trimmed away during theligation step in some other core oligosaccharide-type acceptor molecules has


been suggested for S. enterica serovar Arizonae IIIa [discussed in (236)].However, in the related core structure of E. coli R2, the terminal �-(132)-linkedGlcNAc is present in S-LPS (237), and the corresponding transferase is essentialfor ligation in S. enterica (229). Further experimentation is required to determinewhether this reflects a structural requirement in the ligation-competent acceptoror the need for protein:protein interactions in an assembly complex.

The heterogeneity in LPS molecules isolated from a strain is complex, andmany of the modifications described here may be profoundly affected by growthconditions. It would be particularly interesting to know the extent of heteroge-neity in LPS of bacteria growing within the host.

Phase Variation and the Biosynthesis ofLipooligosaccharide

The biosynthesis of lipooligosaccharide (LOS) in mucosal pathogens representsa special case in terms of structural heterogeneity because many are subject tohigh-frequency phase variation [reviewed in (169, 170)]. The addition of theoligosaccharide extensions in LOS follows essentially the same mechanism usedfor the outer core. Identification of the glycosyltransferases has involved acomparison of LOS structures arising from different mutations [for examples, see(238–242)] as well as overexpression of the appropriate enzymes and analysis oftheir activities using defined acceptors (243–245). In Neisseria (Figure 10),structural studies with an rfaK mutant have provided some insight into the earlyorder of events in synthesis (242). Addition of PEtN residues to the O-3 and O-6positions of the side-branch heptose residue is thought to precede addition of the�-GlcNAc (�-chain) at O-2, the extension of the �-chain on the first heptose, andsubstitution of the glycosyl residue at position O-3 of the side-branch heptose(i.e., �-chain) (240). The addition of the �-chain cannot occur if PEtN alreadyoccupies the O-3 position, so an unidentified PEtN hydrolase has been invoked.The extension of the �-chain requires the prior addition of the �-GlcNAc(�-chain), but because some LOS structures possess the �-chain and lack the�-chain, another processing activity conceivably removes the �-GlcNAc residuelater in the pathway. These putative processing pathways may provide a newdimension in LPS biosynthesis and merit further study.

The precise structure of �-chain produced in the different immunotypes isdetermined by the combination of glycosyltransferases activities expressed fromthe lgt locus (246) (Figure 10). Differential synthesis of LOS structures in bothN. meningitidis and N. gonorrhoeae is complicated by phase variation mediatedby poly(G) tracts in lgtA, C, and D (247–249). The expression of lgtG (�-chain)is regulated in a similar fashion by a poly(C) tract (240). Variations in thenumbers of the Cs or Gs occur during replication, altering the reading frame andleading to premature termination of translation. This seems to be a commontheme in mucosal pathogens, and poly(C) tracts have been implicated in thephase variation in the Lewis antigens presented in the LOS of H. pylori (250,251). The ganglioside GM1-like antigen in C. jejuni LOS is also phase variable


and contains a poly(G) tract (252). A subsequent genomic study of LOS-biosynthesis loci in 11 strains of C. jejuni expressing eight different gangliosidemimics identified poly(G), as well as other genetic mechanisms, resulting in LOSvariation (252a). High-frequency phase variation occurs by a slightly differentmethod in H. influenzae and affects both the carbohydrate structure of the LOSand its modification with PCho substituents, leading to altered LOS immunore-activity and diminished colonization and virulence [for examples, see (253–257)]. In this organism, phase variation is a result of tandem repeats of thesequence 5�-CAAT that lead to slipped-strand mismatches during replication anda translational switch. Collectively, these variations affect the recognition of theLOS structure by the host immune system and can have a significant effect on thebiological properties of the organism.

The addition of sialic acid residues to the �-chain in N. meningitidis LOSinvolves an enzyme (Lst) that can exhibit relaxed specificity (258). Lst from animmunotype L1 isolate can form either �-2,3 or �-2,6 linkages with a terminalgalactose acceptor; in vivo this enzyme generates an �-2,6 linkage. The Lst froman L3 isolate is monofunctional with �-2,3 specificity, but a single amino acidreplacement can convert it into a bifunctional form. This provides an extremeexample of the difficulty and potential pitfalls in assigning activities of trans-ferases from sequence data alone.

STRUCTURE AND BIOSYNTHESIS OFO POLYSACCHARIDES

Structure of O Polysaccharides

Smooth (S)-LPSs are typically produced by isolates of the families Enterobac-teriaceae, Pseudomonadaceae, Pasteurellaceae, and Vibrionaceae, as well as inmany other Gram-negative bacteria. The structural diversity of O antigens isremarkable; more than 60 monosaccharides and 30 different noncarbohydratecomponents have been recognized [reviewed in (259, 260)]. The O-polysaccha-ride repeat unit structures can differ in the monomer glycoses, the position andstereochemistry of the O-glycosidic linkages, and the presence or absence ofnoncarbohydrate substituents. O-repeat units from different structures may com-prise varying numbers of monosaccharides, they may be linear or branched, andthey can form homopolymers (i.e., a single monosaccharide component) or, morefrequently, heteropolymers. In some cases, nonstoichiometric modifications (e.g.,O-acetylation or glycosylation) complicate the identification of a precise O-repeat unit. When the LPS molecules extracted from any S-LPS-containing strainare separated by SDS-PAGE, there is extensive heterogeneity in the sizes of themolecules due to variations in the chain length of the O polysaccharides. This isevident in the classical “ ladder” patter in SDS-PAGE, where each “ rung” up theladder represents a lipid A-core molecule substituted with an increment of one


additional O unit. The spacing between the rungs is determined by the size of theO unit. The S-LPS from most isolates exhibits a preferred and strain-specific sizedistribution pattern (i.e., modal distribution).

The structure of the O polysaccharide defines the O-antigen serologicalspecificity in an organism, but the numbers of unique O antigens within a speciesvary considerably. E. coli, for example, produces �170 O serotypes. In S.enterica, there are 46 serogroups, but modifications to these basal O-repeat unitstructures create many additional “O factors.” O-antigen specificity is onlyrelevant when discussed within a species because there are several exampleswhere all, or part, of an O-repeat unit structure is found in taxonomically distantspecies. A given isolate generally expresses one O antigen, but some bacteriacontain multiple lipid A-core linked polymers, complicating both structuralanalysis and O-antigen terminology. E. coli provides good examples since itslipid A-core can act as an anchor for O antigens, for one form of the Enterobac-terial common antigen (ECA) polymer (261), and for a subset of group 1 andgroup 4 capsular K-antigens [reviewed in (262)]. In some isolates of Salmonella,lipid A-core serves as an acceptor for the classical O antigen as well as theunstable minor antigens T1 and T2 (263). Other isolates of S. enterica producea chromosomally encoded O antigen, together with one resulting from a mobileplasmid carrying a biosynthesis locus (264, 265). P. aeruginosa also expressestwo lipid A-core–linked polymers: a conserved polyrhamnose homopolymerknown as A-band LPS that is analogous to lipid A-core–linked ECA and aserotype-specific O antigen known as B-band LPS [reviewed in (266)]. Insummary, lipid A-core is a versatile anchor for the presentation of cell-surfacepolysaccharides.

The location of O polysaccharide at the cell surface places it at the interfacebetween the bacterium and its environment. Early interest in the chemistry,biosynthesis, and genetics of O polysaccharides was stimulated by their roles asessential virulence determinants and their potential application in vaccine devel-opment, but information is now available for a wide range of species withdifferent lifestyles. The primary role(s) of the O polysaccharides appears to beprotective. In animal pathogens, O polysaccharides may contribute to bacterialevasion of host immune responses, particularly the alternative complementcascade. Assembly of the membrane attack complex is affected by the chemistryof O polysaccharide, its chain length (267), and the relative amounts of longchain S-LPS [reviewed in (268)]. Chain length also affects the sensitivity of E.coli to neutrophil bactericidal/permeability-increasing protein (BPI) (269). In V.cholerae, R-LPS mutants are impaired in colonization of intestinal epithelia,reflecting their increased sensitivity to complement and cationic peptides (270).The presence and chain length of O polysaccharide in Shigella flexneri is alsoessential for invasiveness and subsequent inter- and intracellular spread (271–273) because S-LPS influences the polar localization of the invasion protein(IcsA). The exact role played by O polysaccharide in different bacteria is


therefore variable, and this is potentially magnified in bacteria that can producemore than one lipid A-core-linked polymer.

Biosynthesis of O Polysaccharides

With a few exceptions, the enzymes involved in O-polysaccharide assembly areencoded by genes at the locus historically known as rfb. The majority of systemsare chromosomally encoded, but notable exceptions include the plasmid-encodedO:54 polysaccharide of S. enterica (264, 265). These loci encode enzymes for thesynthesis of novel sugar nucleotide precursors, glycosyltransferases that synthe-size the O polysaccharide, and enzymes required for export processes. Asexpected, the diversity of O-polysaccharide structures is reflected in highlypolymorphic rfb loci. There is a significant literature devoted to the effects oflateral gene transfer, and insertion and deletion events, on the evolution anddiversification of O-antigen types, much resulting from the efforts of P. Reevesand colleagues. [For a detailed survey of the known loci, see the BacterialPolysaccharide Gene Database (BPGD; http: www.microbio.usyd.edu.au/BPGD/default.htm). Most of the rfb operons appear to be constitutively expressed, andthey are often preceded by a JUMPStart/ops sequence indicating that an antiter-mination process is active, as described above for the major core biosynthesisoperon. In one exception, the O-polysaccharide biosynthesis locus of Y. entero-colitica O:3 is transcriptionally regulated by temperature (274, 275), and regu-lation is mediated by a repressor encoded outside of the cluster. The corebiosynthesis genes are not thermoregulated in this organism.

Unlike biosynthesis of the core oligosaccharide, the repeating unit structuresof O polysaccharides are assembled on the membrane-bound carrier, undecapre-nyl phosphate (und-P). This is the same C55-isoprenoid alcohol derivative usedfor synthesis of peptidoglycan and capsular polysaccharides, and L-Ara4Nmodification of lipid A (Figure 5), and it participates in a trans-cytoplasmicmembrane assembly process. O polysaccharides are synthesized from sugarnucleotides by glycosyltransferase enzymes that are often soluble or peripheralmembrane proteins, indicating that assembly of lipid-linked O-repeat unitsoccurs at the inner face of the cytoplasmic membrane. However, O polysaccha-rides are transferred from the carrier lipid and ligated to lipid A-core at theperiplasmic face of the cytoplasmic membrane (276). As a result, the assemblyprocesses must include a mechanism whereby either lipid-linked O polysaccha-rides or lipid-linked O units are delivered to the periplasm. The three currentlyknown pathways for O-polysaccharide biosynthesis are distinguished by theirrespective export mechanisms, and the corresponding characteristic enzymeclasses provide preliminary identification of the pathway used. The pathways arecalled Wzy-dependent, ABC-transporter dependent, and synthase-dependent;each is discussed below. The first two processes are widespread, but the synthasepathway has very limited distribution in O-polysaccharide biosynthesis. Despitethe export differences, the pathways have similar initiation reactions and arecompleted by the same ligation process. This can lead to problems when


heterologous genes are expressed in E. coli K-12 because functions from thehost’s cryptic Wzy-dependent pathway (277) have unexpected interactions withcloned gene products, complicating interpretation of the results (278, 279). Forthis reason, cloning and functional analyses are best done in a K-12 strain suchas E. coli SØ874, where the entire O-polysaccharide biosynthesis locus has beendeleted (280).

Despite the growing body of genetic information for many O-polysaccharidebiosynthesis gene clusters, detailed biochemical investigations are relativelylimited and many of the individual enzymatic mechanisms are often speculativeand based on sequence similarities. The pathways themselves are also repre-sented among systems for the biosynthesis of capsular or exopolysaccharides inGram-negative and Gram-positive bacteria. The O antigens are distinguishedfrom other polysaccharides by the ligation step that links them to lipid A-core,and the translocation machinery required to translocate the completed S-LPSmolecule to the surface of the outer membrane. In the following discussion, wefocus on prototype enzymes for which some biochemical data are available.

Initiation Reactions

The first step in O-polysaccharide biosynthesis involves the formation of anund-PP-linked glycose by transfer of a sugar-1-P residue to und-P. The energy ofthe sugar-phosphate linkage in the donor molecule is conserved in the resultingund-PP-linked intermediate and is available to drive the postpolymerization lipidA-core ligation reaction. The initiating enzymes recognize a hydrophobic lipidcarrier rather than the sugar acceptor used by most other glycosyltransferases.Although there are many different O-repeat structures, the initiation reactions arerelatively well conserved. The best-characterized reactions are catalyzed by theenzymes WbaP (formerly RfbP) from S. enterica (281, 282) and WecA (formerlyRfe) from E. coli (283). WbaP catalyzes the reversible transfer of galactose-1-P(Gal-1-P) from UDP-Gal to und-P and was first characterized in the Wzy-dependent synthesis of O antigens from S. enterica serovars Typhimurium andAnatum (Figure 13). Homologs of WbaP showing glucosyl or galactosyltrans-ferase activity are widespread in capsule and exopolysaccharide synthesis sys-tems. WecA (Rfe) is a GlcNAc-1-phosphate transferase that was firstcharacterized in the Wzy-dependent pathway for ECA synthesis, but it alsoinitiates a variety of Wzy-dependent O polysaccharides from E. coli (284) andShigella species (279, 285). The enzyme appears to have relaxed specificitybecause it can also initiate polymers by the formation of und-PP-GalNAc in someE. coli isolates (286) and perhaps also in Y. enterocolitica O:8 antigen (287). Thistype of relaxed specificity is not confined to WecA; it also occurs in the WbpLenzyme from P. aeruginosa. WbpL shares similarity with WecA and is requiredfor initiation of both A-band and B-band polysaccharides (288). In the formationof B-band, WbpL appears to act as a Fuc2NAc transferase, but availableinformation points to the enzyme catalyzing a different (and as yet unidentified)transfer in the initiation of A-band synthesis.


The Wzy-Dependent Pathway

The classical prototype systems for the Wzy-dependent pathway are the Opolysaccharides from S. enterica serogroups B (Typhimurium) and E1 (Anatum).These O-repeat units have identical Man-Rha-Gal trisaccharide backbones, butthey differ in the presence of a side-branch dideoxyhexose (abequose) substitu-tion in serogroup B and in the linkage between the O-repeat units (2, 5, 9). Earlystudies by M. J. Osborn, H. Nikaido, A. Wright, and P. Robbins and othersestablished the sequence of glycosyl transfer, the required sugar nucleotideprecursors, and the direction of chain growth [reviewed in (7, 10, 263, 289)], andthe corresponding genes have now been identified (290) (Figure 13). Followinginitiation by WbaP, subsequent steps are performed by enzymes that are usuallypredicted to be peripheral membrane proteins. However, there is still no infor-mation concerning the manner in which the various enzymes interact with themembrane and with one another to facilitate their organization into functionalcomplexes.

Following their assembly, the individual und-PP-linked O units are exportedto the site of polymerization at the periplasmic face of the plasma membrane(Figure 13). This process minimally requires a Wzx protein homolog, and thecorresponding structural genes are found in gene clusters for all Wzy-dependentO polysaccharides. In vivo labeling and detection/localization of intermediateshave been used to investigate the effects of a Wzx defect (291). A hybrid strainlacking wzx accumulated und-PP-linked O units that appeared to be located at thecytoplasmic face of the plasma membrane, consistent with the proposal that Wzxis the O unit transporter (“fl ippase” ). However, the small amount of materialinvolved makes definitive localization difficult. Subsequent experiments haveshown that several Wzx homologs from O-polysaccharide biosynthesis systemscan function interchangeably, indicating that Wzx operates independently ofrepeat-unit structure and of initiating sugar (292). Feldman et al. (292) speculatedthat a parallel process may be involved in the transmembrane transfer of

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3Figure 13 Biosynthesis and assembly of O polysaccharides in a Wzy-dependent pathway.Panel A shows the sequence of reactions involved in the formation of the undecaprenyl-linked repeat units and their polymerization in S. enterica serovar Typhimurium. Theindividual glycosyltransferase enzymes are identified in green in the boxes. Panel B showsa model for the events in polymerization. Individual undecaprenyl-linked O-repeat units aretransferred across the membrane by a process involving Wzx (yellow). These intermediatesprovide the substrates for the putative polymerase (Wzy–identified in blue) acting in theperiplasm. Chain extension occurs at the reducing terminus with the nascent chain beingtransferred from its undecaprenyl carrier to the nonreducing terminus of the “new”undecaprenyl-linked subunit. The chain-length modality (i.e., the extent of polymerization)is determined by Wzz (also in blue). The nascent polymer is then ligated to lipid A-core andtranslocated to the outer membrane.


dolichol-linked oligosaccharides in microsomes for glycoprotein biosynthesis. Inan intriguing recent study, the Rft1p protein was discovered to be essential fortranslocation of dolichol-PP-linked intermediates across the endoplasmic retic-ulum membrane for glycoprotein bioysnthesis in the yeast, Saccharomycescerevisiae (292a). Although Rft1-like proteins and Wzx homologues are unre-lated in their primary amino acid sequences, both share distant similarities withpermeases and components of efflux systems, perhaps reflecting a commonfunction in the translocation of their respective glycolipid substrates (M. A.Valvano, unpublished data). Complementation experiments might provide anavenue to determine the extent of the potential functional relationships. Themechanism that flips molecules as large as und-PP-linked O units across the lipidbilayer is unresolved. Predicted Wzx proteins are all highly hydrophobic with 12potential transmembrane domains, and although they share little primarysequence similarity, they do share structural features with bacterial permeases(293). Is Wzx the only protein required for flippase activity? Earlier studiespredicted that the N-terminal domain of the initating WbaP enzyme might alsoplay a role in the flippase activity (294). However, subsequent evidence frommutant derivatives of WbaP (291) and WecA (294a) support the possibility thatthese enzymes might participate in the release of und-PP-linked intermediatesprior to the flippase reaction, in addition to their roles in initiation. Wzx thereforeremains the only known participant in the flippase reaction per se, but furthermechanistic analyses are hampered by difficulties in overexpressing the protein.

At the periplasmic face of the cytoplasmic membrane, und-PP-linked O unitsare polymerized (Figure 13). The reaction involves transfer of nascent polymerfrom its und-PP carrier to the nonreducing end of the new und-PP-linked Orepeat (295, 296). The net effect is chain-length increase by one new repeat unitadded to the reducing end of the nascent polymer. Polymerization is catalyzed bythe O-polysaccharide polymerase, Wzy, and mutants affected in the wzy geneproduce LPS consisting of lipid A-core capped with a single O unit (SR-LPS)(297). This is a defining feature of the pathway. The released und-PP must berecycled to the active monophosphoryl form. Due to the intensive use of und-Pcarriers in the Wzy-dependent biosynthetic pathway, it is characteristicallyinhibited by the antibiotic bacitracin, a compound that inhibits the dephosphor-ylation recycling reaction (298). The O polysaccharides synthesized using theWzy-dependent pathway appear to be exclusively heteropolysaccharides, oftenwith side-branch residues. This mechanism of assembly provides a relativelyefficient process for maintaining the fidelity of synthesis of such complexstructures so its involvement in the synthesis of other cell-surface heteropolysac-charides is perhaps not surprising [reviewed in (262, 289)].

Wzy proteins are all predicted to be integral membrane proteins with 11–13transmembrane domains, and like the Wzx proteins, they exhibit little primarysequence similarity (299, 300). However, Wzy proteins are specific for thecognate O unit or for structures containing the same linkage between O-repeatunits [reviewed in (7, 10, 289)]. The absence of conserved features has compli-


cated the identification of catalytic and binding residues in Wzy; consequently,the mechanism of its activity has not been resolved. For example, it is unclearwhether Wzy is the only component of the biosynthesis machinery required forpolymerization. Since its function has only been studied in vivo, it is not possibleto determine whether contact with other proteins (e.g., Wzx) is also required.Ultimately, resolution of the mechanism of action will require in vitro studies ina reconstituted system, but as with Wzx, such approaches are currently limited bythe difficulty in expressing Wzy in practical amounts (300, a).

The final component of the Wzy-dependent pathway is the Wzz protein(formerly Cld or Rol). The wzz gene was first reported in E. coli and S. entericaserovar Typhimurium (301), but homologs are involved all Wzy-dependentO-polysaccharide biosynthesis systems. Wzz generates the strain-specific modaldistribution of O-polysaccharide chain lengths that is reflected in characteristicclusters of bands in SDS-PAGE [reviewed in (11, 302)]. Models have beenproposed for the underlying process that implicate Wzz, Wzy, and WaaL. Aquantitative analysis of the distribution of chain lengths in the presence andabsence of a functional Wzz determinant led Bastin et al. to conclude that Wzzconfers a preference for chain length on either the polymerase, or the ligase, orboth (303). In the absence of Wzz, the distribution of chain lengths was shownto be independent of chain length, correlating instead with a constant probabilityof transfer to lipid A-core. Wzz was hypothesized to act as a timing clock,interacting with the Wzy polymerase and modulating its activity between twostates that favor either elongation or transfer to the ligase (i.e., chain termination).Morona et al. have suggested that Wzz acts as a molecular chaperone to assemblea complex consisting of Wzy, the WaaL ligase, and und-PP-linked O polysac-charide (304). The specific modality is determined by different kinetics resultingfrom a Wzz-dependent ratio of Wzy to WaaL. A role for WaaL is also inferredfrom the observation that Wzz can only regulate chain length of polymers that arelinked to lipid A-core [reviewed in (11)]. In some cases, bacteria have more thanone wzz gene. P. aeruginosa has two chromosomal wzz genes (266), and in S.flexneri, the chromosomal wzz gene product is accompanied by a plasmid-encoded homolog (305). The biological effect of possessing two Wzz activitiesis unknown.

Localized regions of Wzz primary sequences are relatively well conserved, asare the predicted secondary structures [reviewed in (11, 302)]. Wzz homologs arelocated in the cytoplasmic membrane, and all have two transmembrane helicesflanking a periplasmic loop with a predicted propensity for coiled-coil structure(302). The ability of Wzz to somehow modulate the kinetics of the polymeriza-tion and/or ligation reactions suggests that the protein must recognize and forma complex with either Wzy, or WaaL, or both. The coiled-coil domains may beimportant for such interactions, and oligomers of Wzz have been identified bychemical cross-linking experiments (306). However, there is currently no defin-itive evidence of cross-linking of Wzz to either Wzy or WaaL. The function ofWzz homologs is not restricted by a specific O-repeat unit structure. Attempts to


define specific regions required for Wzz modality have involved expression ofheterologous wzz genes, comparative sequence analyses, construction of hybridproteins, and site-directed mutagenesis of specific residues (306–308). To date,no clear picture has emerged concerning the regions of the protein that conferspecificity.

The ABC-Transporter-Dependent Pathway

This pathway is currently confined to linear (unbranched) O-polysaccharidestructures and involves chain extension by processive addition of glycosylresidues to the nonreducing terminus of the und-PP-linked growing chain. Thereis no requirement for a specific polymerase enzyme, and the polymer is com-pleted at the inner face of the cytoplasmic membrane. Export of the polymer tothe outer face for ligation requires an ABC (ATP-binding cassette) transporter,precluding the involvement of Wzx.

To date, the O polysaccharides formed by this pathway are all initiated by ahomolog of WecA (Figure 14). In the characterized pathways, the resultingund-PP-GlcNAc acts as a primer for chain extension, and while the GlcNAcresidue will be transferred to lipid A-core during ligation, this residue occurs onlyonce per chain. This role contrasts to the involvement of WecA in the Wzy-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 14 Biosynthesis and assembly of O polysaccharides in an ABC-transporter-dependent pathway. Panel A shows the predicted structures of undecaprenyl-linked inter-mediates in the biosynthesis of the O9a antigen of E. coli and the D-galactan I polymerfound in several serotypes of K. pneumoniae. The glycosyltransferase enzymes involved ineach step are indicated below the structures. Biosynthesis is broken down into the formationof a common primer (shown in red), addition of an adaptor region, chain extension of therepeat unit, and addition of a chain terminator (for details see the text). In the O9a structure,the chain is thought to be terminated by addition of 3-O-methylmannose, but the enzymeresponsible has not been identified. In D-galactan I, the structure of the chain terminator hasnot been established. Chain elongation occurs by processive glycosyl transfer to thenonreducing terminus. Several enzymes in these pathways are bifunctional. In the case ofthe galactosyltransferase WbbO, both of its activities are required for adaptor synthesis, butonly one (WbbO2) participates in chain extension. Panel B provides a model for thetrans-membrane assembly system. The glycosyltransferases are shown in green. TheABC-transporter formed by Wzm and Wzt is required for transfer of the undecaprenyl-linked polymer to the periplasmic face of the membrane, where it is ligated to lipid A-coreand translocated to the outer membrane. It is presumed that the polymer remains attachedto the undecaprenyl carrier throughout the export process. While chain extension and exportare separable by mutations in the ABC-transporter, it is conceivable that the two processesare temporally coupled in vivo. Within the nascent polymer, the primer/adaptor is identifiedby the red-filled hexagon, the residues of the repeating-unit domain by black-filled circles,and the chain terminator by the open hexagon.


dependent pathway where the enzyme is required for each lipid-linked O repeat,and GlcNAc is therefore found within the repeat unit structure itself. Theinvolvement of WecA is well established for the polymannose O polymers of E.coli O8, O9, and O9a (and their structural counterparts in K. pneumoniae O5 andO3) and for the galactose-containing O polysaccharides (D-galactan I) from K.pneumoniae. Transfer of GlcNAc-1-P has been unequivocally identified as theinitial reaction in vivo for E. coli O8 (309), and this correlates with thedemonstrated activity of the enzyme in ECA synthesis as well as the preferredreaction in vitro (310). Furthermore, definitive structural analysis of the core Opolysaccharide linkage region in K. pneumoniae O3 and O5 identifies theGlcNAc residue resulting from WecA (235, 235a; E. Vinogradov, E. Fridrich,L. L. MacLean, M. B. Perry, O. Petersen, C. Whitfield, manuscript in prepara-tion). In the next step in biosynthesis, an O-polysaccharide-specific glycosyl-transferase adds the adaptor between the und-PP-GlcNAc primer and the repeatunit domain and commits the lipid intermediate to the chain extension pathway.In E. coli, O9 adaptor formation involves the addition of a single mannoseresidue by WbdC (formerly MtfC) (310) (Figure 14). As with the WecA-mediated transfer, this reaction occurs only once per chain, and the residue isevident in the linkage regions for K. pneumoniae O3 and O5 LPSs (235, 235a;E. Vinogradov, E. Fridrich, L. L. MacLean, M. B. Perry, O. Petersen, C.Whitfield, manuscript in preparation). In the biosynthesis of D-galactan I, thecorresponding activity is mediated by the bifunctional galactosyltransferase,WbbO. Both activities of WbbO are involved in formation of the adaptor, but onealso participates in subsequent chain extension reactions (311, 312) (Figure 14).In the chain extension phase, the repeating-unit domain is assembled by proces-sive transfer of residues to the nonreducing terminus of the und-PP-linkedacceptor (310, 312, 313). The transferases involved in the elongation reaction canbe monofunctional or, as in the case of the E. coli O9 and O9a polymannans, canadd two or more residues of a given linkage type (310, 314, 315). This may beuseful for maintaining the fidelity of O-repeat unit structure that may be definedby the linkage distribution rather than by different monomers. Overall thepathway requires the participation of only one und-P molecule per polymerchain, and so und-PP recycling is not a limiting factor and the process is resistantto bacitracin (298).

Following polymerization, the nascent O polysaccharide must be exportedacross the cytoplasmic membrane for ligation. Studies with Y. enterocoliticaserotype O:3 first identified pathway mutants that accumulated intracellular Opolysaccharide (316), but the same phenomenon has been described in K.pneumoniae O1 (317) and P. aeruginosa A-band LPS synthesis (318). In thesesystems, surface assembly requires an ABC transporter encoded by the O-poly-saccharide biosynthesis cluster. The genes for the transporters were initiallyidentified by protein sequence similarities shared with the ABC-2 subfamily ofATP-binding cassette transporters (319). ABC-2 transporters consist of an inte--gral membrane protein with an average of six membrane-spanning domains, and


a hydrophilic protein containing an ATP-binding motif or Walker box. The genesfor these two components have now been identified in a number of O-poly-saccharide biosynthesis clusters. As with other ABC-transporters involved intransmembrane export, the membrane-spanning (Wzm) homologs for O-poly-saccharide biosynthesis generally exhibit little primary sequence identity buthave nearly identical hydropathy plots. In contrast, primary sequences of theATP-binding (Wzt) homologues are much more highly conserved, particularly(and predictably) in the nucleotide-binding region.

Little is known about the organization and exact mode of action of ABC-transporters for O polysaccharides, and unlike MsbA (see above), no structuraldata are available. Much of the information is inferred from studies andhypothetical models with comparable systems involved in group 2 capsulesynthesis in E. coli and other bacteria [reviewed in (320)]. Other ABC-trans-porters, such as the Sec system for protein export [reviewed in (321)], have beenparticularly influential. In the group 2 capsule systems, the ABC-transporterforms part of an assembly complex that may allow polymerization and exportprocesses to proceed in a coordinated manner [reviewed in (262)]. The exportprocess is driven by ATP hydrolysis, and the Wzt protein may undergo anATP-binding-induced conformational change, resulting in its insertion into themembrane via an interaction with the Wzm, introducing polymer into the channel(Figure 14). A series of insertion-deinsertion events would then drive thepolymer through the membrane. Experiments that directly address this hypoth-esis are difficult to formulate. Since E. coli K-12 can ligate to its lipid A-corepolymers from any of the three known assembly pathways, we assume that thematerial is presented to the K-12 ligase in the same form (i.e., attached tound-PP). If this is indeed the case, the export machinery must handle und-PP-linked polymer.

The ABC-transport dependent pathway does not involve a Wzz chain-lengthdeterminant, yet S-LPSs in bacteria with the pathway do exhibit strain-specificmodal distributions (11). In one extreme example, the Aeromonas salmonicida Opolysaccharides have a uniform chain length (322). The observation that modal-ity is conserved when O polysaccharides are expressed from cloned genes inheterologous hosts (i.e., E. coli K-12) demonstrates that modal regulation is afunction of one or more of the components encoded by the biosynthesis clusteritself. The fact that chain-length distribution is altered when Wzm/Wzt areexpressed at higher levels than the glycosyltransferases (317) favors a competi-tion between the termination-export and chain-extension activities, but whatterminates the processive chain-extension process? An intriguing feature ofseveral O polysaccharides from ABC-transporter-dependent pathways is thepresence of novel nonreducing terminal residues on the glycan chains. In thepolymannan O8 glycans of E. coli (and the K. pneumoniae O3 and O5 counter-parts), the chains end in a 3-O-methylmannose residue (E. Vinogradov, E.Fridrich, L. L. MacLean, M. B. Perry, O. Petersen, C. Whitfield, manuscript inpreparation; 235, 235a, 323). 2-O-methyl groups are found at the termini of V.


cholerae O1 chains, and these are associated with the seroconversion to Inabafrom Ogawa type (324, 325). Recent structural studies have identified �-Kdo and�-Kdo at the termini of the K. pneumoniae O4 and O12 antigens (polymers withno Kdo residues in their repeat units), respectively (E. Vinogradov, E. Fridrich,L. L. MacLean, M. B. Perry, O. Petersen, C. Whitfield, manuscript in prepara-tion; 235, 235a). In the ABC-transporter-dependent O polysaccharide of B.bronchiseptica (326), a unique 2,3,4-triamino-2,3,4-trideoxy-�-galacturonamidederivative is found at the chain terminus (327). It therefore appears that terminalmodifications are a widespread phenomenon in this pathway, and it is temptingto speculate that they provide an essential component of the ABC-transporter-dependent systems. The frequency by which these residues are incorporatedcould alter the assembly complex from a chain-elongation to a chain-terminationmode, initiating the export process. Detailed analysis of mutants lacking theterminal modifying enzymes should provide insight into the mechanism(s), andcandidates have been identified (B. R. Clarke & C. Whitfield, unpublishedresults).

The Synthase-Dependent Pathway

Synthases are processive glycosyltransferases that have the capacity to synthe-size polymers within a single polypeptide. Included among their members areenzymes involved in biosynthesis of cellulose and chitin. Other well-documentedexamples occur in the biosynthesis of capsular polysaccharides including hyal-uronic acid by Streptococcus pyogenes and other bacteria [reviewed in (328)],and the type 3 capsules of S. pneumoniae (329, 330). These enzymes have twoconserved domains. Domain A is a region found in both processive andmonofunctional �-glycosyltransferase enzymes, whereas domain B is confined tothe processive examples. Chain extension occurs at the nonreducing terminus(330, 331), but the identity of the lipid carrier on which the nascent chains areextended (331a), and the process of initiation, have not been resolved for thebacterial capsule synthases.

The plasmid-encoded O:54 antigen of S. enterica serovar Borreze is currentlythe only known example of a synthase-dependent O polysaccharide (265, 332,333). Synthesis of the poly-N-acetylmannosamine homopolymer that comprisesthe O:54 antigen is initiated by WecA (265) (Figure 15). The first ManNAcresidue commits the und-PP-GlcNAc primer to O:54 biosynthesis and is trans-ferred by the nonprocessive ManNAc transferase WbbE (formerly RfbA) (334).The first two steps in this pathway are therefore reminiscent of those in theABC-transporter–dependent process. The second transferase encoded by theO:54 biosynthesis cluster, WbbF (formerly RfbB), is a member of the HasA(hyaluronan synthase) family of enzymes, and it is proposed that this enzymeperforms the chain-extension steps. Synthases are integral membrane proteinswith similar topology (332, 335) and are believed to catalyze a vectorialpolymerization reaction, simultaneously extending the polysaccharide chain andextruding the nascent polymer across the plasma membrane (328, 332). As with


the ABC-transporter–dependent pathway, the product of the synthase pathway isligated to lipid A-core by the E. coli K-12 ligase (265). Although this isconsistent with the possibility that the exported form of the polymer is und-PP-linked, there is no information that casts light on the exact mechanism of export.Also unknown is the process involved in chain termination. Studies on biosyn-thesis of the streptococcal type 3 capsule have led to a proposal that chaintermination in that synthase results from an abortive translocation step in whichthe normal passage of nascent polymer between sites that add new residues isdisrupted (330). When one of the sugar nucleotide donors for the type 3 polymer(which has a disaccharide repeat unit) is limiting, chain termination is favored,followed by release of the nascent polymer from the enzyme. While a veryinteresting proposal, firm conclusions are complicated by the uncertainty sur-rounding the exact mechanism of glycosyltransfer by the synthases. Earlyspeculation about a two-glycosyltransfer site model has been challenged by morerecent structural data for �-glycosyltransferases [reviewed in (226, 227, 336)].

Seroconversion Reactions

The impact of nonstoichiometric substitutions on O antigenicity is widelyrecognized. The phenomenon is well studied in Salmonella and Shigella, whereadditions of acetyl groups or glucose residues create novel O factors. In manycases, the modifying enzymes are carried by lysogenic or cryptic bacteriophages.

The oafA gene product encodes the O-acetyltransferase conferring O:5 spec-ificity in Salmonella (337). Other well-characterized O-acetyltransferases includeOac, encoded by the Shigella seroconverting bacteriophage SF6 (338), and theO-acetyltransferase carried by P. aeruginosa bacteriophage D3 (339). Acetyl-CoA provides the donor for O-acetylation, and early experiments suggested thatthe acceptor for the O-acetyltransferase reaction is the und-PP-linked O unit[discussed in (263)]. The location of the reaction and the precise extent ofacetylation of individual O-repeat units have not been definitively established.The use of acetyl-CoA as the initial donor suggested a cytoplasmic location, butrecent analysis suggests the possibility of a periplasmic reaction [reviewed in(340)]. In general, O-acetyltransferases fall within four families. The largermembers such as OafA are integral membrane proteins and contain an N-termi-nal domain that shares some similarity with human acetyl-CoA transporter. It isproposed that these enzymes export acetate and acetylate the acceptor. Soluble(and putatively extracellular) O-acetyltransferases are accompanied by an inte-gral membrane protein (a member of the AlgI family) that is proposed to exportacetate for the modification reaction. Further experimentation is now required toaddress these interesting hypotheses. An extracellular location would allow theuse of acceptors comprising either und-PP-O repeat or und-PP-O-polysaccharide.

The donor for nonstoichiometric glucosyl substituents in S. enterica Opolysaccharides is an unusual lipid intermediate, �-glucosylmonophosphorylun-decaprenol (und-P-Glc) (341–343), resembling the donor for the L-Ara4N resi-dues that modify lipid A (Figure 5). In serogroups B and D, glucosylation is a


postpolymerization reaction and is assumed to occur at the level of the und-PP-linked O polysaccharide (341, 343, 344). In contrast, in serogroups C1 and C2,glucosylation is reported to occur before polymerization, at the level of individ-ual und-PP-linked O units (345), and these contradictory data need to be revisitedwith additional experiments. Glucosylation is particularly prevalent among the Oantigens of Shigella where the genetic determinants have been described[reviewed in (346)]. Each Shigella glucosylation locus contains three genes andis contained within DNA from a lysogenic or cryptic phage. Homologues arerecognized in the sequence of the Salmonella bacteriophage P22 and in a crypticprophage from E. coli. Preliminary functional assignments of the genetic deter-minants have been made (347, 348). The GtrB enzyme appears to be a GT thatsynthesizes und-P-Glc by transferring glucose from UDP-glucose to und-P. Aserotype-specific transferase (Gtr) is responsible for the transfer of the glucoseresidue from the und-P-Glc donor to the acceptor. The gtrA gene encodes anintegral membrane protein that is proposed to serve as a flippase to transferund-P-Glc to the periplasmic face of the membrane, where glucosylation mayoccur. Whether this protein operates alone or in conjunction with other O-poly-saccharide biosynthesis machinery (e.g., Wzx) is unknown. As expected, the gtrAand B genes are interchangeable among systems, but the gtr genes are specific fora given modification.

Another well-documented bacteriophage-encoded modification involves achange in the specificity of the Wzy polymerase, resulting in an alteration in theanomeric linkage between O-repeat units in the final polymer. The classicalexample of this modification is the �15-mediated seroconversion in Salmonella(349), but recent detailed insight into the process has been provided by studieswith the D3 lysogenic seroconverting phage of P. aeruginosa. The D3 lysogenacetylates the host O polysaccharide as well as directing an � to � switch in the

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 15 Biosynthesis and assembly of O polysaccharides in a synthase-dependentpathway. The only current example is the plasmid-encoded O:54 antigen of S. entericaserovar Borreze. Panel A shows the predicted structure of the undecaprenyl-linked inter-mediate. The primer is made by WecA. The WbbE enzyme then adds a single ManNAcresidue as an adaptor, but the precise linkage has not been determined. The synthase(WbbF) is then required for chain extension, generating a repeat-unit domain withalternating �(1,3) and �(1,4) linkages. Growth is at the nonreducing terminus. Panel Bshows a model for the transmembrane assembly process, with glycosyltransferases identi-fied in green and putative transport functions in yellow. Available data for this system, andinformation for other bacterial synthases, are consistent with the conclusion that WbbFserves as a processive glycosyltransferase with dual linkage specificity, as well as anexporter that moves undecaprenyl-linked intermediates to the periplasm. While chainextension and export are shown as separate processes, they are possibly temporally coupledin vivo. The nascent polymer is then ligated to lipid A-core and translocated to the outermembrane.


linkage between O repeats (339). The end result is a seroconversion fromserotype O5 to O16. The D3 phage carries a �-specific Wzy but also encodes ahighly hydrophobic 31-residue �-polymerase inhibitor (Iap) that inactivates the“normal” chromosomal Wzy protein. The �15-seroconversion also involves aputative small polypeptide inhibitor (350). The relationships (if any) between theinhibitors have not been established, and more work is required to unravel thefascinating mechanism by which they act.

The examples above all involve modifications to a Wzy-dependent pathway,but there is evidence for similar and often nonstoichiometric glycosylation andO-acetylation modifications in ABC-transporter pathways too. For example, anumber of Klebsiella O serotypes express galactose-containing O polysaccha-rides based on a common D-galactan I backbone but differ in modifications to thisstructure (351–357). The O-polysaccharide biosynthesis loci only contain genesfor D-galactan I synthesis (317, 358, 359), and the location(s) of the genesresponsible for the modifications has not been identified yet, so mechanisticinformation is not available.

EXPORT OF LPS TO THE CELL SURFACE

The method by which completed LPS molecules are translocated to the cellsurface represents one of the most interesting open issues in LPS biosynthesis.The LPS molecules must pass across the periplasm and through the outermembrane. The translocation processes do not discriminate between LPS withdifferent O-polysaccharide structures, and S-LPS and R-LPS molecules areapparently translocated with equal efficiency (360). The targeting of LPS to theouter membrane is predicted to require a pathway, rather than being mediated bystructural features of the LPS molecule itself.

It has long been known from work in Salmonella that new LPS moleculesappear on the cell surface at a limited number of sites (361, 362). Membrane-adhesion sites, regions where the inner and outer membranes come into apposi-tion, were implicated. These structures are only visualized by specific electronmicroscopy and have been controversial [for discussion, see (289, 363, 364)].However, current models for the assembly and translocation of capsular poly-saccharides do invoke a molecular “scaffold” across the periplasm comprisingproteins involved in the translocation process, and experimental evidence lendssupport to the hypotheses [reviewed in (262, 320)]. The Type II and III proteinsecretion systems in bacteria offer similar examples of multiprotein translocationmachineries (365). Membrane-adhesion sites may reflect points where the trans-location machinery effectively creates continuity between a biosynthesis com-plex and the outer membrane, providing an organized domain for thetranslocation of macromolecules.

The capsule biosynthesis loci from Gram-negative bacteria encode proteinsinvolved in translocation across the periplasm and outer membrane, but no


corresponding proteins are encoded by the LPS core or O-polysaccharidebiosynthesis loci. Translocation systems for LPS potentially involve generalizedouter membrane assembly functions in the cell. Unlike the capsules, LPS areessential for growth in the laboratory, and so mutations affecting dedicatedtranslocation components would be lethal, limiting the isolation of informativemutants from random screens. While there are enough unknowns in the bacterialgenomes to potentially accommodate translocation functions, other well-knownsystems may also participate in the process. For example, the TolA protein hasbeen implicated in the processing or translocation of S-LPS in E. coli (366). Thefour-protein Tol system is required for import of bacteriocins and bacteriophagesacross the outer membrane. E. coli K-12 tolA mutants are viable, but in awild-type strain making S-LPS, a tolA mutation, is lethal. One intriguinginterpretation of this finding is that the translocation systems contain essentialcomponents needed for all LPSs, and additional components specifically requiredfor S-LPS. This finding may provide a useful starting point for resolution of thetranslocation pathways.

FUTURE DIRECTIONS FOR LPS RESEARCH

As is apparent from the discussion of the biosynthesis of LPS, detailed mecha-nistic understanding of the biosynthesis of the core and O polysaccharide regionslags behind the lipid A area. One reason for this is undoubtedly the structuralvariation of these regions of the LPS molecule and the corresponding diversity ofthe underlying enzymes. However, genomic and other sequence-based studieshave shown that, despite the variations in details, the overall pathways fromdifferent bacteria are quite conserved. This will allow the development of broadlyrepresentative prototype systems and a focused approach to the elucidation ofcritical enzymes. The mechanistic details can now be addressed. Clearly, themost intriguing questions in lipid A and LPS assembly involve the transport ofthe various LPS components across the cytoplasmic membrane and translocationof the completed molecules to the cell surface. Examination of these potentiallymultienzyme processes is experimentally challenging. However, recent progresswith similarly complex systems in protein export and secretion points to possibleexperimental avenues and provides confidence that these processes will beresolved in time. The development of well-defined in vitro enzymatic systems forO-polysaccharide polymerization and ligation to lipid A-core would serve as auseful prelude to biochemical studies of LPS export and assembly into the outermembrane.

To ensure substantial progress, an interdisciplinary experimental approachwill be needed in future studies of LPS endotoxins. The application of structuralbiology, bacterial genomics, and animal knockout models to LPS biology is stillin its infancy. These newer strategies need to be combined with older approaches,like enzymology, carbohydrate chemistry, and membrane biochemistry. The


remarkable images provided by the X-ray structures of the LpxA trimer (51), theouter-membrane protein FhuA bound to a single LPS molecule (128), and theMsbA lipid A/phospholipid transporter (120) hint at what the future has in store.Novel antibiotics, vaccines, and antiinflammatory agents are likely to emergefrom an in-depth understanding of LPS-protein interaction at the atomic level.

ACKNOWLEDGMENTS

Research in the laboratory of C. R. H. Raetz is supported by NIH GrantsR01-GM-51,310 and R37-GM-51,796. C. Whitfield is recipient of a CanadaResearch Chair, and his research in this area is funded by the Natural Sciencesand Engineering Research Council and the Canadian Bacterial Diseases Network(NCE program). We thank all the members of our laboratories for their criticalcomments and stimulating discussions. This review is dedicated to ProfessorLaurens Anderson of the University of Wisconsin-Madison.

The Annual Review of Biochemistry is online at http://biochem.annualreviews.org

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