JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Feb. 1999, p. 973–980 Vol. 181, No. 3

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Effect of wzx (rfbX) Mutations on A-Band and B-BandLipopolysaccharide Biosynthesis in

Pseudomonas aeruginosa O5LORI L. BURROWS AND JOSEPH S. LAM*

Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 15 July 1998/Accepted 17 November 1998

The wbp cluster of Pseudomonas aeruginosa O5 encodes a number of proteins involved in biosynthesis of theheteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz,the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in the wbp cluster, ispredicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondarystructure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or “flippase.” Insertionof a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-bandlipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successfulgeneration of chromosomal wzx gene replacement mutations. Surprisingly, inactivation of wzx also led to amarked delay in production of the ATP-binding cassette–transporter-dependent, D-rhamnose homopolymer,A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans.WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band andB-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol.Microbiol. 28:1103–1119, 1998). These results suggest that the delay in A-band LPS production may arise frominsufficient access to WbpL when the completed B-band O unit is not successfully translocated to theperiplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of wzx mutants appeared tohave accumulated second-site mutations which either restored the normal expression of A-band LPS orabolished A-band expression completely. Complementation studies showed that all of the additional mutationsaffecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

Current models explaining the biosynthesis and assembly oflipopolysaccharide (LPS) O antigens invoke two separate path-ways, termed the Wzy (Rfc)-dependent and the Wzy-inde-pendent–ATP-binding cassette (ABC)–transporter-depen-dent pathways (reviewed in reference 36). Wzy is the O-anti-gen polymerase and is involved in the biosynthesis of hetero-polymeric O antigens. Two other proteins required in the Wzy-dependent pathway are Wzx (RfbX), the putative O-antigenunit translocase or “flippase,” and Wzz (Rol, Cld), the regu-lator of O-antigen chain length (36). Individual O-antigensugar units are synthesized on the isoprenoid lipid carrier un-decaprenol phosphate (C55P) at the cytoplasmic face of theinner membrane. Following synthesis, individual O-antigenunits are thought to be translocated by an integral membraneprotein, Wzx, to the periplasmic face of the cytoplasmic mem-brane, where they are polymerized by Wzy (25, 36). The chainlength of the growing heteropolymer is controlled by the Wzzprotein (3, 4, 7, 28) via an unknown mechanism. The hetero-polymer is then covalently attached to the previously synthe-sized core lipid A by the O-antigen ligase, encoded by waaL(rfaL) in Escherichia coli and Salmonella enterica serovar Ty-phimurium.

In contrast, homopolymeric O antigens appear to be synthe-sized processively on C55P without an O-antigen polymerase(36). Most homopolymeric O antigens are transported via atwo-component, ABC-type transporter, which moves the as-sembled homopolymer across the cytoplasmic membrane (19,

29, 36). Once the homopolymer has been translocated to theperiplasmic face of the cytoplasmic membrane, it can be li-gated to core lipid A by the O-antigen ligase. Recently, analternative transport mechanism for the O:54 homopolymer ofSalmonella enterica serovar Borreze was proposed, in which theO antigen is assembled and transported across the cytoplasmicmembrane in the absence of an ABC transporter (18).

Pseudomonas aeruginosa simultaneously produces two formsof LPS, called A-band and B-band LPS. A-band LPS containsa neutral homopolymer of a-D-rhamnose (23). The A-bandbiosynthetic cluster has been cloned and sequenced and hasbeen shown to contain genes encoding a typical two-compo-nent transporter system (29). B-band LPS is the O-antigen-containing form and is a heteropolymer of di- to pentasaccha-rides, containing uronic acids and very rare sugars, such aspseudaminic acid (20). The wbp cluster encoding the biosyn-thesis of the B-band O antigen of serotype O5 has been clonedand sequenced (6, 7, 24). It resembles other gene clustersencoding the synthesis of heteropolymeric O antigens in that itcontains wzx, wzy, and wzz homologues. The wzy and wzz genesfrom serotype O5 have previously been characterized in ourlab (7, 9). The start codon of the putative wzx gene (formerlywbpF) overlaps the stop codon of wzy (6). Analysis of thededuced amino acid sequence of Wzx showed that it is ahydrophobic protein with multiple membrane-spanning do-mains in its predicted secondary structure, similar to that ofother Wzx proteins. Homologues of wzx have been identified inmany bacterial species in both LPS O-antigen and capsularbiosynthetic clusters (37). While the primary sequence homol-ogies of Wzx from different bacteria can be poor, the proteinsshare similar secondary structures. Wzx has not been exten-sively studied, probably due to obstacles encountered during

* Corresponding author. Mailing address: Department of Microbi-ology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.Phone: (519) 824-4120, ext. 3823. Fax: (519) 837-1802. E-mail: jlam@uoguelph.ca.

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the cloning of the gene in isolation and creation of null mu-tants (25, 26). Chromosomal mutations in wzx have been de-scribed as deleterious and difficult to study (31). However, Liuand coworkers (25) were able to use a plasmid-encoded O-anti-gen cluster carrying a nonpolar transposon insertion in wzx todemonstrate that wzx mutants appear to accumulate O-antigenunits on the cytoplasmic face of the inner membrane.

In this study, we examined the function of Wzx in P. aerugi-nosa O5 through the creation of chromosomal wzx knockoutmutants. As far as we are aware, this is the first report of thesuccessful generation of such mutants. Analysis of these knock-out strains confirmed the involvement of Wzx in B-band LPSbiosynthesis and helped define the interrelationship betweenthe Wzy-dependent and the Wzy-independent pathways of O-antigen biosynthesis in P. aeruginosa O5.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains and plasmids used in thisstudy are listed in Table 1. P. aeruginosa strains were grown either on Pseudo-monas isolation agar (Difco), Luria broth and agar (Sigma), or Davis minimalmedium (Fisher). E. coli strains were grown on Luria broth and agar (Sigma).Where necessary, antibiotics (all from Sigma) were added as described previ-ously (9).

DNA manipulations. Chromosomal DNA was isolated from P. aeruginosausing the method of Goldberg and Ohman (13). Plasmid DNA was isolated byusing the Qiagen midi-prep or mini-plasmid kits (Qiagen Inc.) as directed by themanufacturer. Restriction and modification enzymes were used as directed bythe manufacturers.

Plasmids were introduced into E. coli by CaCl2 transformation (17) and intoP. aeruginosa by electroporation using a Bio-Rad (Richmond, Calif.) GenePulser apparatus following the manufacturer’s protocols. Electrocompetent cellsof P. aeruginosa were prepared by growing the cells to mid-log phase in Luriabroth and then washing the cells twice for 5 min each in sterile 10% roomtemperature glycerol followed by immediate resuspension in the same solution.Cells were either used immediately or frozen at 280°C for future use. Plasmidswere also introduced into P. aeruginosa by biparental mating with E. coli SM10carrying mobilizable plasmids of interest (34).

Creation of isogenic chromosomal knockout mutants. The gene replacementstrategy of Schweizer and Hoang (33) was used for the creation of knockoutmutations in wzx as described previously (8, 9). In the event that isolates wereobtained in which only single crossover events had occurred, these merodiploidswere plated overnight at 37°C on modified Luria medium containing 5% sucrose

and no NaCl (38). This treatment selects for cells which have lost the sacB-containing vector DNA following a double crossover event that generates truerecombinants. Correct gene replacement was ascertained through Southern blotanalysis of chromosomal DNA isolated from gentamicin-resistant, sucrose-sen-sitive, carbenicillin-sensitive mutants.

A similar strategy was used to create a double knockout mutant. An AL (forA late) wzx strain, S2, was used as the background for the introduction via elec-troporation of a copy of wbpM (6) inactivated by a nonpolar carbenicillin resis-tance cassette, and cloned into the sacB-containing suicide vector pEX18Tc(pFV169-18TcCb) (16). Correct gene replacement was ascertained by Southernblot analysis of chromosomal DNA from gentamicin-resistant, carbenicillin-re-sistant, sucrose-sensitive, tetracycline-sensitive mutants.

Southern blot analysis. Restriction-enzyme-digested chromosomal DNA wasseparated on 0.8% agarose gels, transferred to Zetaprobe nylon membrane(Bio-Rad) by capillary transfer, and crosslinked to the membrane using a Strata-linker (Stratagene, La Jolla, Calif.). For detection of specific fragments, probeDNA was labelled with digoxigenin-dUTP (Boehringer Mannheim, Laval, Que-bec, Canada), and hybridization and detection were performed according to themanufacturer’s directions.

SDS-PAGE and Western immunoblot analysis. LPS from P. aeruginosa wasprepared by the method of Hitchcock and Brown (15). The LPS preparationswere separated on standard discontinuous sodium dodecyl sulfate (SDS)–12%polyacrylamide gels and visualized by silver staining using the method of Dubrayand Bezard (11). For immunoblotting, LPS separated by SDS-polyacrylamide gelelectrophoresis (PAGE) was transferred to nitrocellulose (5). Nitrocelluloseblots were blocked with 3% skim milk followed by overnight incubation withhybridoma culture supernatants containing monoclonal antibody (MAb) MF15-4(specific for O5 B-band LPS) (21) or MAb N1F10 (specific for A-band LPS) (22).A goat anti-mouse F(ab9)2-alkaline phosphatase second antibody conjugate(Jackson Immunoresearch, West Grove, Pa.) was used to detect the first anti-body. The blots were developed using a substrate containing 0.3 mg of Nitro BlueTetrazolium/ml and 0.15 mg of BCIP (5-bromo-4-chloro-3-indolylphosphate to-luidine)/ml in 0.1 M bicarbonate buffer (pH 9.8).

Time course experiments. Growth curves of strains PAO1, S2, and X10showed that there were no significant differences in the rates of growth of theparent and mutant strains (not shown). To demonstrate the appearance ofA-band LPS over time, the mutant and parent strains were grown in 50 ml ofLuria broth at 37°C with 200-rpm shaking. Aliquots of 0.5 ml were removedbeginning at 12 h after inoculation (early stationary phase) and then at 6- to 12-hintervals up to 60 h postinoculation, and the optical densities at 600 nm of thesamples were determined. The cells were harvested and used to prepare LPS viathe method of Hitchcock and Brown (15). The LPS was analyzed by SDS-PAGEand Western immunoblotting as outlined above.

Nucleotide sequence accession number. The corrected DNA sequence of thewzx gene is available from GenBank under accession no. U50396.

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype, phenotype, or properties Reference or source

P. aeruginosaO5 Strain PAO1, wild type A1 B1 15O5 wzxs S2 PAO1, wzx insertion mutation at SstI; AL B2 This studyO5 wzxs S7 PAO1, wzx insertion mutation at SstI; A1 B2 This studyO5 wzxx X10 PAO1, wzx insertion mutation at XhoI; AL B2 This studyO5 wzxx X6 PAO1, wzx insertion mutation at XhoI; A2 B2 This studyO5 wzxx X14 PAO1, wzx insertion mutation at XhoI; A2 B2 This studyO5 wzxx X24 PAO1, wzx insertion mutation at XhoI; A1 B2 This study

E. coliJM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Dlac-proAB F9[traD36 proAB1 lacIq lacZDM15] 39SM10 thi-1 thr leu tonA lacY supE recA RP4-2-Tc::Mu, Kmr 35

PlasmidspUCP26 4.9-kb pUC18-based broad-host-range vector; Tcr 36pEX100T Gene-replacement vector; oriT1 sacB1; Apr 34pUCGM Source of Gmr cassette; Apr Gmr 31pAK1900 4.75-kb pGEM3Zf(1)-based vector with pRO1600 oriR, Apr A. KropinskipFV155T 5.2-kb HindIII insert containing wzy, wzx, hisHF, wbpG blunt-cloned in SmaI site of pEX100T This studypFV155TG pFV155T with Gmr cassette in unique SstI site within wzx This studypFV162-26 3.1-kb BamHI-BglII insert containing wzx, hisHF in pUCP26 under lac promoter 6pFV162-26G pFV162-26 with Gmr cassette in unique XhoI site within wzx This studypFV162-26TG Insert of pFV162-26G blunt cloned into SmaI site of pEX100T This studypFV110 1.4-kb HindIII-XbaI insert containing wbpL in pAK1900 8pFV114 10.5-kb SpeI insert containing wzx through wbpL in pAK1900 This study

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RESULTS AND DISCUSSION

Characterization of Wzx. In a previous study (6), we report-ed Wzx (formerly WbpF) to be 316 amino acids (aa) long, alength inconsistent with those of other Wzx proteins, whichrange from approximately 400 to 500 aa. Reanalysis of theDNA sequence in the region containing wzx showed the actualsize of the wzx gene to be 1,236 bp. This open reading frame ispredicted to encode a protein of 411 aa, in agreement with thesizes of other Wzx proteins.

Repeated attempts to express Wzx by both in vivo and invitro methods were not successful, although the HisH and HisFproteins encoded immediately downstream of wzx (Fig. 1) andon the same recombinant plasmid (pFV162-26) were readilyexpressed (6). Our observations are consistent with previousreports that the high hydrophobicity and presence of rare ormodifying codons within the coding regions for Wzx and Wzyproteins make them extremely difficult to express using cur-rently available methods (9, 26, 27).

Analysis of chromosomal wzx mutants. In order to demon-strate the function of Wzx in P. aeruginosa O-antigen biosyn-thesis, a nonpolar Gmr cassette was used to insertionally inac-tivate the wzx gene of serotype O5. The first set of wzx mutantsconstructed had a gentamicin cassette inserted at a unique SstIsite located 462 bp from the 39 end of the gene (wzxs; Fig. 1).However, there were concerns that insertion of the Gmr cas-sette near the 39 end of the gene may have led to the genera-tion of a truncated but potentially functional peptide. Thisconsideration prompted the construction of a second set of wzxmutants. The second group, wzxx, had the nonpolar Gmr cas-sette inserted in a XhoI site 200 bp from the 59 end of wzx (Fig.1). In our hands, the yield of both types of wzx mutants com-pared to that obtained for other P. aeruginosa LPS genes usingthe same methodology was very low (6, 7, 9, 29, 30). The pooryield is consistent with the reported difficulties encountered byothers during attempts to isolate such mutants (25, 26). Cor-rect insertion of the gentamicin resistance cassette within wzxin both sets of mutants was confirmed by Southern immuno-blot analysis (Fig. 2).

On silver-stained SDS-polyacrylamide gels, the gentamicin-resistant wzxs mutants were devoid of B-band LPS (Fig. 3).Interestingly, of 21 wzxs mutants generated, 20 also lacked theladder-like banding pattern typical of A-band LPS on silver-stained SDS-polyacrylamide gels. This result was confirmed

using MAb N1F10, which is specific for A-band LPS (Fig. 3).This is the first instance in which mutation of a wzx gene hasbeen shown to affect synthesis of a Wzy-independent, homo-polymeric polysaccharide. Similar results were obtained for thewzxx set of mutants (not shown).

Production of A-band LPS is delayed in wzx mutants. Dur-ing analysis of the wzxs mutants, we noted that LPS prepara-tions made from fresh overnight plates contained no detect-able A-band LPS on silver-stained SDS-polyacrylamide gels orWestern immunoblots. In contrast, preparations made fromplates that were several days old appeared to have substantialamounts of A-band LPS. This delay in A-band LPS productionwas reproducible and occurred in cultures grown on solid me-dia (both Luria agar and Pseudomonas isolation agar) as wellas in broth. Strains which displayed this phenotype were des-ignated AL to distinguish them from those which were trulyA2.

Analysis of LPS production by AL wzxs mutants over timecompared with the parent strain PAO1 was performed. Com-parison of the growth rates of the parent and a representativeAL wzxs mutant (S2) showed no significant differences (notshown). While the parent strain had substantial amounts ofboth A- and B-band LPS after 12 h of growth, the S2 mutantproduced only rough LPS (core-lipid A) with no detectable Aband or B band (Fig. 4). After 18 to 24 h, A-band LPS becamedetectable in the preparations from the S2 culture (Fig. 4). Incontrast, no B-band LPS could be detected at any time duringthe experiment.

As mentioned above, our concern that production of a trun-cated but partially functional Wzx protein by wzxs mutants wassomehow responsible for the AL phenotype was addressedthrough generation and analysis of the wzxx series of mutants.Analysis of the LPS produced by wzxx mutants on silver-stainedSDS-polyacrylamide gels showed that of 39 mutants obtained,none produced B-band LPS, and the majority produced littleor no A-band LPS after 12 h of growth. Results from a timecourse experiment similar to the one described above showedthat, again, the amount of A-band LPS produced by a repre-sentative wzxx mutant (X10) increased over time from unde-tectable to substantial (Fig. 4).

Analysis of atypical A1 or A2 wzx mutants. In contrast to theAL wzx mutants described above, a subset of wzx mutants (1of 21 wzxs and 4 of 39 wzxx mutants) produced substantial

FIG. 1. Physical map of locations of plasmids used in this study with respect to the B-band LPS gene cluster. The individual open reading frames within the wbpcluster of serotype O5 are shown as arrows at the top of the figure. The wzx gene described in this study is shown in black, while the wbpL and wbpM genes are shownin grey. The positions of the two individual Gmr cassette insertions within the wzx gene are shown as black triangles. Mutants with an insertion at SstI are designatedwzxs, while mutants with an insertion at XhoI are designated wzxx. B, BamHI; B2, BglII; H, HindIII; N, NruI; S, SalI; Ss, SstI; X, XhoI; Xb, XbaI. For clarity, only selectedrestriction sites are shown.

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amounts of A-band LPS during all phases of growth (repre-sentative mutants are shown in Fig. 5). In addition, at least 3 of39 wzxx mutants produced no A-band LPS at all, even uponprolonged incubation (Fig. 5). These two types of wzx mutantswere designated A1 and A2, respectively. Despite the differ-ence in A-band phenotype among the wzx mutants, Southernblot analysis of representative strains showed no gross rear-rangements in ;5 kb of DNA encompassing the site of theinsertional mutation (Fig. 2). The synthesis of A-band LPS byboth the AL (after prolonged growth) and the A1 wzx mutantsconfirmed that Wzx is not directly necessary for A-band LPSsynthesis. Therefore, the mere lack of Wzx could not explainthe deficiency in A-band LPS biosynthesis in AL (during earlygrowth phases) and A2 wzx mutants.

The delay in A-band LPS expression in AL strains wasthought to be due to the reduction in some componentrequired for production of both types of LPS, stemming fromthe interruption in B-band LPS synthesis. A-band and B-bandLPS have clearly been shown to be synthesized by differentpathways (7, 9, 29). However, there are components shared byboth pathways. Recently, we showed that WbpL, which ini-tiates synthesis of B-band LPS by transferring N-acetyl-6-de-oxygalactosamine-1-P (Fuc2NAc-1-P) to C55P (6), is also re-

quired to initiate the synthesis of A-band LPS (30). WbpL is ahomologue of E. coli WecA (Rfe), an enzyme that initiates thesynthesis of enterobacterial common antigen and a variety of Oantigens through the addition of N-acetylglucosamine-1-P(GlcNAc-1-P) to C55P. In the case of homopolymeric O anti-gens, including A-band LPS, the residue added by WecA-WbpL acts solely as a primer and does not become part of theO repeat unit (36). WbpL and WecA were both able to initiatethe synthesis of A-band LPS in a wbpL::Gmr mutant ofP. aeruginosa O5, likely through the addition of GlcNAc-1-P toC55P (30).

We postulated that the interruption of B-band biosynthesisafter formation of the O-antigen unit, but prior to its translo-cation, might in some way affect the availability of WbpL. Areduction in the availability of WbpL would hinder the ini-tiation of A-band polymer synthesis, generating the AL phe-notype. Alternatively, the introduction of errors or rear-rangements in the putative operon wbpG-wbpL, following therecombination events required to generate the knockout mu-tant, could have polar effects on the expression of wbpL. Thelatter prospect is less likely, since a number of individual wzxmutants with an AL phenotype, each presumably arising fromunique recombination events, were isolated. In addition, South-ern blot analyses of the wzy-wbpG region in which the recom-bination events occurred show that there are no gross rear-rangements (Fig. 2), suggesting that polar effects are unlikelyto be the cause of the AL phenotype.

The growth rates of AL wzx mutants (which do not appear tohave acquired additional mutations affecting LPS biosynthesis)do not seem adversely affected (not shown), as one might ex-pect of cells with insufficient free C55P to support normal pep-tidoglycan synthesis. However, based on the potentially dele-terious nature of wzx mutations, it is possible that thosemutants eventually isolated already contain compensatory mu-

FIG. 2. Southern blot analysis of selected wzx mutants. Chromosomal DNAfrom representative AL (S2 and X10) and A1 (S7 and X24) wzx mutants from thewzxs and wzxx series and a representative A2 (X14) wzxx mutant was digestedwith HindIII and separated on a 0.8% agarose gel, transferred to a nylon mem-brane, and probed with a dUTP-digoxigenin-labelled BamHI-BglII fragmentcorresponding to the insert of pFV162-26. All mutants show an increase in thesize of the HindIII fragment of approximately 0.9 kb, corresponding to the sizeof the Gmr cassette. No gross rearrangements that could be responsible for thevaried A-band LPS phenotypes of these mutants were found in this region.

FIG. 3. Analysis of LPS from a representative AL wzxs mutant, S2. LPSisolated from 12-h cultures of the AL wzxs mutant S2 and from the O5 parentstrain was analyzed with silver-stained SDS-polyacrylamide gels as well as byWestern immunoblotting with LPS-specific antibodies. The mutant produced nodetectable A-band or B-band LPS. Complementation with pFV162-26 (wzx,hisHF; Fig. 1) restored both A- and B-band LPS biosynthesis.

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tations that permit them to grow in the presence of the wzxmutation.

The delay in A-band LPS production is alleviated by sup-plying WbpL in trans. To test the hypothesis that the availabil-ity of WbpL was limiting in AL cells, we transformed AL wzxsmutant S2 with a high-copy-number plasmid carrying the se-rotype O5 wbpL gene under the control of the lac promoter(pFV110). If insufficient WbpL is available in AL cells, provi-sion of excess WbpL in trans should mitigate the delay inA-band LPS expression. Analysis of LPS from S2 transformedwith pFV110 (wbpL) showed that the presence of the plasmidrestored the ability of S2 to produce A-band LPS without delay(Fig. 6). Interestingly, the A2 wzx mutant randomly chosen forfurther analysis (X14) appears to have a secondary mutationaffecting wbpL, since it was rendered A1 by the addition ofpFV110 (wbpL) alone (see below).

From these data, it appears that A-band biosynthesis per seis not impeded by the wzx mutation and that WbpL is ex-pressed and functional in AL wzx mutants, since A-band LPScan eventually be detected. Typically, some WbpL molecules

would initiate A-band LPS biosynthesis; however, the fre-quency of the initiation event likely relies upon the number ofWbpL molecules available. Possible explanations for the AL

phenotype include a reduction in the amount of wbpL expres-sion in wzx mutants, a decrease in the normal rate of initiationof A-band LPS, possibly due to the presence of B-band O unitson C55P, or interference with normal WbpL function or avail-ability. We are currently analyzing the transcription of thewbpG-wbpL operon in both PAO1 and S2 to explore the firstpossibility. However, provision of wbpL expressed in multicopyfrom an unregulated promoter could overcome any of thesedifficulties.

Complementation analysis of wzx A1 and A2 mutants. Wethought that emergence of A1 or A2 derivatives of wzx mu-tants could be due to the selection for strains with spontaneoussecondary mutations in genes necessary for LPS biosynthesis,perhaps due to a reduction in free C55P. Liu and coworkers(25) showed that wzx mutants appeared to accumulate a singleO-antigen unit on C55P. In P. aeruginosa, interruption of B-band biosynthesis after formation of the O-antigen unit, but

FIG. 4. Analysis of the production of A-band LPS over time. (A) LPS was harvested from the O5 parent strain and from the AL mutant wzxs S2 as described inMaterials and Methods and analyzed on silver-stained SDS-polyacrylamide gels. While the parent strain (O5) produced substantial amounts of both A- and B-bandLPS after 12 h of growth, the S2 mutant produced no perceptible amounts of either LPS after 12 h of growth. After 24 to 36 h of growth, the mutant produced sufficientA-band LPS to be detectable on silver-stained SDS-polyacrylamide gels. (B) Western immunoblot analysis of LPS from the AL wzx mutants S2 (wzxs) and X10 (wzxx)using LPS-specific MAbs. In comparison with 12-h cultures of the parent strain (O5), which contain both A- and B-band LPS, 12-h cultures of S2 and X10 contain nodetectable A- or B-band LPS. A-band LPS is detectable after 18 to 24 h of growth, while no B-band LPS could be detected over the duration of the experiment. Theeffect of both mutations in wzx appears to be the same.

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prior to its translocation, may cause C55P to be sequestered.Removal of C55P from the cellular pool would be deleteriousfor other cell functions, such as peptidoglycan formation. Thestrong pressure to overcome the reduction in availability ofC55P may lead to accumulation of second-site mutations. Themost likely sites for such mutations would be in the B-bandO-antigen genes or in housekeeping pathways which feed intoLPS synthesis in order to prevent formation of the B-band Ounit altogether. For example, a second-site mutation in theB-band cluster would prevent formation of the O-antigen uniton C55P, allowing the synthesis of A-band LPS to continuenormally (A1 cells) in the presence of the wzx mutation (unlessthey occurred within wbpL itself).

In support of this hypothesis, a number of wzx mutants withA-band LPS expression atypical of a wzx mutant were isolated.This subset included five A1 wzx mutants and at least three A2

wzxx mutants, examples of which are shown in Fig. 5. The in-ability to complement either A1 or A2 wzx mutants usingpFV162-26 (wzx) alone, a construct that rendered isogenic AL

wzx strains A1B1, confirmed that A1/A2 strains had likelyaccumulated additional mutations leading to loss of expressionof A band, B band, or both.

Complementation of the atypical strains with the entire geneclusters necessary for either A- or B-band biosynthesis identi-fied the B-band LPS genes as the site of secondary mutations.Introduction of pFV3 (23), carrying the A-band LPS biosyn-thetic genes, into the A2 wzx mutant X14 was not able torestore A-band biosynthesis, locating its secondary mutation(s)elsewhere (Fig. 7). In contrast, pFV100 (6, 24) carrying the wbp(B-band) gene cluster could restore B-band LPS biosynthesisin both A1 and A2 wzx mutants as well as A-band synthesis inthe wzx A2 mutant (Fig. 7). These results imply that, in thesemutants, a secondary mutation(s) affecting LPS biosynthesiswas in the B-band genes. Further analysis showed that the A2

wzx mutant X14 could be complemented to A1B1 by pFV114(Fig. 1), which contains both wzx and wbpL, and to A1 but notB1 by pFV110, containing wbpL (Fig. 7). Taken together,these results show that X14 contains the original wzx mutationas well as a secondary mutation in wbpL.

Generation of double knockout mutants. We attempted toreplicate the phenotype of A1 wzx mutants by introducing a

second knockout mutation of the B-band LPS biosyntheticgenes into an AL wzx mutant, S2. We reasoned that preventionof synthesis of the first sugar of the O unit, Fuc2NAc, wouldprevent accumulation of any material on C55P. The highlyconserved gene, wbpM, that lies at the 39 end of the B-bandLPS gene cluster is implicated in Fuc2NAc biosynthesis (6).The wbpM gene was inactivated with a nonpolar carbenicillinresistance cassette, and the resulting construct was introducedinto the chromosome of the AL wzx mutant, S2.

The PAO1 parent strain, the AL wzx strain (S2), and the S2wbpM::Cbr mutants were grown for 12 h (the time point atwhich S2 had no detectable A-band LPS; Fig. 4). LPS wasprepared by the method of Hitchcock and Brown (15) andexamined on silver-stained SDS-polyacrylamide gels and West-ern immunoblots with LPS-specific MAbs. Introduction of thewbpM::Cbr mutation was not able to relieve the AL phenotypeof the S2 mutant (not shown). This result may mean that theatypical A1 and A2 wzx mutants did not arise from an AL wzxbackground. Alternatively, since the function of WbpM inFuc2NAc biosynthesis has not yet been ascertained, it may notbe the appropriate target for inactivation in order to recreatethe A1 wzx phenotype from an AL wzx mutant.

With the exception of ABC-type transporters (19, 29), theidentification and analysis of components of the LPS transportmachinery have proven to be complicated. Problems related tothe low copy number and integral membrane location of trans-port proteins and the deleterious effects on cell growth causedby their mutation (31) have hampered elucidation of this as-pect of LPS biosynthesis. Despite the precedent, we have suc-cessfully generated wzx chromosomal knockout mutants anddid not experience the reported difficulties encountered duringattempts to clone wzx in the absence of other LPS genes (26).

P. aeruginosa is a unique model system in which two forms ofLPS with independent pathways of biosynthesis are copro-

FIG. 5. Analysis of LPS from atypical wzx mutants. LPS from representativewzx mutants which either produced A-band LPS without delay (wzxs mutant S7and wzxx mutant X24) or produced no A-band LPS even after prolonged growth(wzxx mutants X6 and X14) was analyzed on silver-stained SDS-polyacrylamidegels and Western immunoblots using LPS-specific MAbs. The LPS from the O5parent strain, S7, and X24 was prepared from 12-h cultures, while the LPS fromthe X6 and X14 cultures was prepared from 36-h cultures. None of the mutantsmade B-band LPS at any point during growth.

FIG. 6. Alleviation of the AL phenotype by wbpL in trans. The AL wzxsmutant S2 was transformed with pFV110, a high-copy-number plasmid contain-ing wbpL (Fig. 1). Analysis of LPS from 12-h cultures of the transformantsshowed that they were producing A-band LPS without delay, suggesting WbpLwas limiting in S2.

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duced. The only analogous system that has been studied inenterics involves the concomitant production of a homopoly-meric O8 or O9 O antigen with a heteropolymeric lipid A-core-linked “capsular” exopolysaccharide in E. coli (called KLPS) (2,10, 12). Although a wzx homologue has been identified in theK40 cluster of E. coli O8 and both polymers are WecA depen-dent (2), wzx mutants are not yet available (1). Therefore, it isnot clear whether a wzxK40 mutation would affect production ofthe O8 or O9 O antigens.

This study has demonstrated that wzx is essential in the syn-thesis of the heteropolymeric B-band O antigen of P. aerugi-nosa O5 and that its mutation can affect the synthesis of thehomopolymer, A band. In addition, the key role of WbpL ininitiation of both A- and B-band synthesis has been reempha-sized. By generating chromosomal wzx mutants, we have laidthe foundation for understanding the role of Wzx in translo-cation of O units and the effect of wzx mutations on the activ-ities of the initial glycosyltransferase, WbpL.

ACKNOWLEDGMENTS

Funding for the study was provided to J.S.L. from the MedicalResearch Council of Canada (grant MT14687). L.L.B. is the recipientof a Canadian Cystic Fibrosis Foundation Fellowship.

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