Identification of three oligo-/polysaccharide-specific ligasesin Yersinia enterocoliticammi_7918 125..136

Elise Pinta,1,2 Zhilin Li,1 Julia Batzilla,3

Maria Pajunen,4 Tiina Kasanen,1 Kamila Rabsztyn,1,5

Alexander Rakin3 and Mikael Skurnik1,6*1Department of Bacteriology and Immunology, HaartmanInstitute, University of Helsinki, Helsinki, Finland.2Department of Medical Biochemistry and Genetics,University of Turku, Turku, Finland.3Max von Pettenkofer-Institute,Ludwig-Maximillians-University, Munich, Germany.4Department of Biosciences, Division of Biochemistryand Biotechnology, University of Helsinki, Helsinki,Finland.5Department of Microbiology, University of Silesia,Katowice, Poland.6Helsinki University Central Hospital LaboratoryDiagnostics, Helsinki, Finland.

Summary

In lipopolysaccharide (LPS) biosynthesis of Gram-negative bacteria the lipid A-core oligosaccharide(LA-core) and O-polysaccharide (O-PS) biosynthesispathways proceed separately and converge in peri-plasmic space where the waaL-encoded ligase joinsO-PS onto LA-core. Enterobacterial common antigen(ECA) biosynthesis follows that of O-PS except thatECA is usually ligated to phosphatidylglycerol (PG)and only rarely to LA-core. In Yersinia enterocoliticaserotype O:3 LPS is composed of LA-inner core (IC)onto which a homopolymeric O-PS, a hexasaccharidecalled outer core (OC), and/or ECA are ligated. Wefound that an individual O:3 LPS molecule carrieseither OC or O-PS substitution but not both. Related tothis, we identified three genes in Y. enterocolitica O:3that all expressed O-PS ligase activity in the Escheri-chia coli DwaaL mutant. The LPS phenotypes ofY. enterocolitica O:3 single, double and triple ligasemutants indicated that two of ligases, named asWaaLos and WaaLps, had a preferred substrate speci-ficity for OC and O-PS, respectively, although withsome promiscuity between the ligases; the third ligasenamed as WaaLxs was not involved in LPS or ECA

biosynthesis. In Y. enterocolitica O:8 the WaaLos

homologue (Ye1727) ligated a single pentasaccharideO-unit to LA-IC suggesting that in both Y. entero-colitica O:3 and O:8 WaaLos is an oligosaccharide(OS)-specific ligase. Finally, Yersinia pestis andY. pseudotuberculosis carry only the waaLps gene,while either waaLos or waaLxs or both are additionallypresent in other Yersinia species. This is the firstreport on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.

Introduction

Lipopolysaccharide (LPS) biosynthesis in Gram-negativebacteria takes place in two converging entities (Raetz andWhitfield, 2002). The lipid A-core oligosaccharide (LA-core) synthesis initiates in the cytoplasm and continues atthe cytoplasmic leaflet of inner membrane (IM). The LApartis formed first onto which the core is built by dedicatedglycosyltransferases that sequentially transfer the sugarresidues from nucleotide diphosphate (NDP)-activatedsugar molecules. The completed LA-core is translocated tothe outer leaflet of the IM by a flipping mechanism. Thesynthesis of the second entity, the O-polysaccharide(O-PS, also known as O-antigen), takes place directly at IMwhere dedicated glycosyltransferases transfer sequen-tially the sugar residues onto the carrier lipid, undecapre-nylphosphate (Und-P). Two main types of O-PS arerecognized: (i) heteropolymeric O-PS composed of oli-gosaccharide (OS) repeat units and synthesized by theO-unit polymerase (Wzy)-dependent pathway and (ii)homopolymeric O-PS formed of a repeating monosaccha-ride and synthesized by the ABC transporter-dependentpathway. In the Wzy-dependent pathway a single O-repeatunit, and in the ABC transporter-dependent pathway afull-length homopolymeric O-PS, is synthesized on Und-P;when complete, both structures are translocated by aflipping mechanism to face the periplasmic space (Raetzand Whitfield, 2002) where, in the Wzy-dependentpathway, most Und-P-P-O-units are polymerized by Wzywith the help of chain length determinant Wzz to Und-P-P-O-PS. Finally, the Und-P-P-carried OS or O-PS is recog-nized by a dedicated ligase, WaaL, that transfers it fromUnd-P-P to the LA-core (Raetz and Whitfield, 2002; Ruizet al., 2009). The completed LPS molecules are thereafter

Accepted 2 November, 2011. *For correspondence. E-mail mikael.skurnik@helsinki.fi; Tel. (+358) 9191 26464; Fax (+358) 9191 26382.

Molecular Microbiology (2012) 83(1), 125–136 � doi:10.1111/j.1365-2958.2011.07918.xFirst published online 28 November 2011

© 2011 Blackwell Publishing Ltd

translocated to the outer leaflet of outer membrane(OM) by a recently identified ABC transporter-dependentpathway (Malinverni and Silhavy, 2009; Ruiz et al., 2009).

WaaL is an integral IM protein having a distinct topologywith 12 transmembrane (TM) segments and a relativelylarge periplasmic loop between TM segments 9 and 10(Islam et al., 2010). WaaL has been identified from anumber of Gram-negative organisms including Salmo-nella enterica, Pseudomonas aeruginosa, Escherichiacoli, Klebsiella pneumoniae, Helicobacter pylori, Edward-siella tarda and Vibrio cholerae (Schild et al., 2005;Fresno et al., 2006; Perez et al., 2008; Berry et al., 2009;Hug et al., 2010; Islam et al., 2010; Xu et al., 2010). As arule, a single waaL gene is present in these organisms.

Yersinia enterocolitica O:3 is a Gram-negative bacte-rium expressing LPS composed of LA-inner core (IC) ontowhich are linked via a glycosidic bond a homopolymericO-PS, a hexasaccharide known as outer core (OC), and/oran enterobacterial common antigen (ECA) (Radziejewska-Lebrecht et al., 1998; Skurnik, 1999; 2003; 2004). Thelatter, however, is more commonly linked via a phosphodi-ester bond to phosphatidylglycerol (PG) (Kuhn et al., 1988;Rick et al., 1998; Kajimura et al., 2006). Of these substitu-tions, only the glycosidic linkage of OC to LA-IC is known(Radziejewska-Lebrecht et al., 1998; Pinta et al., 2009),those of O-PS and ECA have not yet been resolved due tomethodological and technical difficulties. A distinct char-acteristic in Y. enterocolitica O:3 is that both O-PS andOC are linked to LA-IC and this has made it possible toconstruct mutants expressing different combinations ofO-PS and OC: for example, strain YeO3-R1 expresses OCbut no O-PS (Al-Hendy et al., 1992), strain YeO3-c-OCexpresses O-PS but no OC while strain YeO3-c-OC-Rlacks both OC and O-PS (Biedzka-Sarek et al., 2005).Furthermore, different parts of LPS function as receptorstructures for bacteriophages, e.g. OC is recognized byfR1-37 and O-PS by fYeO3-12 (Pajunen et al., 2000;Kiljunen et al., 2005). Both O-PS and OC are immunogenicand monoclonal antibodies have been raised against thesestructures (Pekkola-Heino et al., 1987; Biedzka-Sareket al., 2005).

The biosynthesis of the OC hexasaccharide takes placesimilar to the Wzy-dependent pathway of O-PS biosynthe-sis, i.e. onto the Und-P-P carrier lipid (Skurnik et al., 1995;1999) while that of the homopolymeric O-PS uses theABC transporter-dependent pathway (Zhang et al., 1993).Similar to other Gram-negative bacteria the polysaccha-ride biosynthetic genes of Y. enterocolitica O:3 involved inOC, O-PS and ECA biosyntheses are organized into geneclusters (Zhang et al., 1993; Skurnik et al., 1995; Rabsztynet al., 2011). The OC and O-PS gene clusters, when intro-duced on a plasmid to heterologous hosts such as Yersiniapestis, Y. enterocolitica serotype O:8 or E. coli, allow theY. enterocolitica O:3 OC or O-PS, respectively, to be pro-

duced as a part of the LPS of the constructed strains(Al-Hendy et al., 1991; Kiljunen et al., 2005; 2011). TheOC, O-PS and ECA gene clusters contain 9, 8 and 12genes, respectively, including genes for biosynthesis ofNDP-sugar precursors, glycosyltransferases and translo-cation of the Und-P-P-carried OC, O-PS or ECA to peri-plasmic space (Zhang et al., 1993; Skurnik et al., 1995;Rabsztyn et al., 2011). However, the gene clusters do notencode a ligase that would transfer the OC, O-PS or ECAfrom the Und-P-P-carrier to LA-core.

In this work we set out to identify the OC ligase ofY. enterocolitica O:3 but discovered three different ligase-encoding genes in contrast to one normally present inother Gram-negative organisms. Based on their preferredsubstrate specificities we named two of the genes aswaaLos and waaLps. We demonstrated that WaaLos ismainly responsible for the OC ligation and WaaLps, for theO-PS ligation. The third ligase was not involved in eitherLPS or ECA biosynthesis, therefore we named it asWaaLxs.

Results and discussion

Our original goal in this work was to identify the OC ligaseinvolved in the Y. enterocolitica O:3 LPS biosynthesis. Wehypothesized that an OC-ligase mutant would not expressOC; thus mutants lacking OC would be resistant andwould survive selection with enterocoliticin, a bacteriocinthat uses OC as its receptor (Strauch et al., 2003). Usingthis strategy, we screened a Cat-Mu transposon libraryconstructed in strain YeO3-R1 (see Supporting informa-tion for details) and isolated several enterocoliticin-resistant OC-lacking mutants. To our disappointment,sequence analysis of the Cat-Mu-insertion sites of thesemutants revealed no OC-ligase candidates; however, weidentified several where Cat-Mu insertion had interruptedgenes involved in LPS biosynthesis. For example, weisolated deep-rough strains with transposon insertionsin genes that are involved in IC biosynthesis codingfor putative glycosyltransferases (waaF, waaC, waaE)or enzymes involved in biosynthesis of NDP-sugar-precursors (galU) (Fig. S1). In addition, a number ofmutants had transposon insertion in the rfaH gene. RfaH isa transcription elongation factor that binds specifically tooperon polarity sequence (ops) and that together withother factors increase the processivity of the RNA poly-merase complex such that transcription proceeds overlong distances (Bailey et al., 1997). As OC genes areorganized in a 13-kb cluster that is preceded by the JUMP-start sequence (Hobbs and Reeves, 1994; Skurnik et al.,1995) our results indicated that RfaH identifies the opselement within the OC gene cluster JUMPstart sequenceand is absolutely required for efficient expression of theOC cluster genes.

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Identification of a ligase candidate

In mutant YeO3-R1-M089 (Fig. S1) the Cat-Mu transposonwas inserted into a gene annotated as icd coding forisocitrate dehydrogenase, one of the enzymes of the citricacid cycle. Icd should not be directly involved in OC bio-synthesis. The icd gene in Y. enterocolitica O:8 strain 8081is ye1725 that is a first gene in a putative operon (Fig. 1A).This raised a possibility that enterocoliticin resistancecould be due to a polar effect on a downstream gene.Similarity searches and prediction of membrane topology

indicated that the gene ye1727 product had qualities thatcould be associated with a ligase. Although its sequenceidentity with the E. coli WaaL was low (17%), its size of 412amino acids and its predicted TM segment topology wasidentical to those predicted with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) to known ligases, i.e.it had 12 TM segments and a large periplasmic loopbetween TM segments 9 and 10 (Perez et al., 2008; Islamet al., 2010). A search of the (at that time partial) genomicsequence data of Y. enterocolitica serotype O:3 strain Y11(Batzilla et al., 2011) showed that a full homologue of

Fig. 1. The gene organization of the ligase-encoding loci in Y. enterocolitica serotype O:3 and O:8 strains Y11 and 8081 based on genomicsequences (database Accession No. AM286415 and FR729477 respectively). The genes are indicated by arrows and the annotated CDSsidentified above (for O:3) or below (for O:8) the genes. The ligase-encoding genes are shaded.A. The waaLos loci. The O:3 locus compared with that of O:8 contains an insertion that has integrated into the icd gene as the 3′-end of thegene is duplicated in O:3. The homologous genes between the genomes are indicated by shaded rectangles. The waaLos gene appears to beorganized into an operon with an upstream gene (ye1726 in O:8) encoding an unknown GT-2 family glycosyltransferase. The Cat-Mutransposon insertion in strain YeO3-R1-M089 was within the icd gene.B. The waaLps loci are identical in O:3 and O:8 and the waaQ (encoding heptosyltransferase III) and waaLps genes appear to form an operon.The lsrK and lsrR genes are part of an autoinducer-encoding gene cluster extending to the right of the picture. The arcD and arcA geneproducts are involved in arginine metabolism.C. The waaLxs loci are identical in O:3 and O:8, and the gene is preceded by ye0778, a conserved hypothetical protein present also inY. pestis and Y. pseudotuberculosis, and followed by mutS/fdv gene encoding a DNA mismatch repair protein. The mrpA, mrfB and mrfCgenes are part of a fimbriae-encoding operon.

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ye1727 was present in Y. enterocolitica O:3 genome. Wedecided to carry out a more detailed study of this gene wenamed waaLos (see below).

In the course of this work, a putative waaL gene inY. pestis (ypo0417) had been identified (Anisimov et al.,2010). The ypo0417 homologue in the Y. enterocolitica O:8genome is ye0532; and a homologue was also identifiedfrom the genomic sequence of the O:3 strain Y11 encodinga protein of 414 residues (Fig. 1B). We named this gene aswaaLps (see below). Finally, in a BLASTP search we identi-fied a third ligase candidate gene (ye0777) from Y. entero-colitica O:8 genome encoding a protein with c. 50% identityto the O:3 WaaLos and WaaLps sequences. A homologue tothis gene named as waaLxs was also found from thegenomic sequence of Y. enterocolitica O:3 Y11 (Fig. 1C),encoding a 554-amino-acid protein. For both of these latterligase candidates the TMHMM program predicted also a TMsegment topology characteristic for ligases (Perez et al.,2008; Islam et al., 2010) although WaaLxs is predicted tocontain in the C-terminus a c. 100-amino-acid-long extracytoplasmic fragment. While we had no experimental evi-dence for the predicted TM topologies and it has beenshown that the predictions may produce misleading results(Islam et al., 2010) the fact that all three WaaL candidatesgave similar ligase-specific TM predictions encouraged usto characterize their roles as ligases.

All three ligase candidates complement in E. coli theDwaaL phenotype

To reveal whether the identified genes encode ligase activ-ity we introduced the waaLos, waaLps and waaLxs genes ofY. enterocolitica O:3 into DwaaL E. coli strain CLM24/pMF19 using plasmids pLos, pLps and pLxs (Table 1)respectively. The waaL-deficient strain CLM24/pMF19(Feldman et al., 2005) carries plasmid pMF19 encoding arhamnosyltransferase (WbbL) that allows for the comple-tion of the O16 subunit synthesis in E. coli K-12 strains (Liuand Reeves, 1994; Feldman et al., 1999). The LPS phe-notypes of the in trans complemented strains were analy-sed by DOC-PAGE and Western blotting. In all strains therestoration of E. coli O:16 O-PS expression was demon-strated (Fig. S2) confirming that the waaLos, waaLxs andwaaLps genes of Y. enterocolitica O:3 encoded ligase activ-ity in E. coli background.

The waaL mutants of Y. enterocolitica O:3

To characterize their roles in the LPS biosynthesis weconstructed single, double and triple waaL mutants ofY. enterocolitica O:3 (Table 1). To simplify the recognitionof the mutants, the strain designations contain abbrevia-tions of the mutated genes as follows: Los for waaLos,Lps for waaLps and Lxs for waaLxs. For example, thewaaLos strain is YeO3-c-Los, the waaLos waaLps double

mutant is YeO3-c-Los-Lps, and the triple mutant isYeO3-c-Los-Lps-Lxs.

The single ligase mutant phenotypes of Y. enterocoliticaO:3

While the OC and O-PS phenotypes of the wild type andthe single mutant strains YeO3-c-Lps and YeO3-c-Lxswere almost identical (Fig. 2, lanes 1, 5 and 6, Table 2),YeO3-c-Los in silver-stained DOC-PAGE and in Westernblotting with monoclonal antibodies (mAb) 2B5 and TomA6(Figs 2 and 3) showed strongly reduced amounts of bothOC and O-PS (Fig. 2, lane 4, Fig. 3, lane 3). At the sametime a strong IC band became visible in DOC-PAGE. TheOC phenotype of YeO3-R1-Los was identical to that ofYeO3-c-Los (Table 2). The OC and O-PS phenotypes werealso assessed by sensitivity of the bacteria to OC-specificfR1-37 and enterocoliticin, and to O-PS-specific bacte-riophage fYeO3-12 (Table 2). In line with the result that afaint OC band was detected from the YeO3-c-Los bacteriathe mutant demonstrated strongly reduced sensitivity tofR1-37 and was enterocoliticin-resistant (Table 2). Thissuggested that the residual O-PS on YeO3-c-Los couldblock the reduced number of OC molecules from entero-coliticin but not from fR1-37. In line with this, the YeO3-R1-Los bacteria were killed by enterocoliticin although muchless efficiently than the parental strain YeO3-R1 (Table 2).

A likely explanation to the residual OC on YeO3-c-Losand YeO3-R1-Los could be the promiscuity of the threedifferent ligases expressed in Y. enterocolitica O:3. Thus,in the absence of the specific OC ligase WaaLos, one orboth of the other two ligases could (albeit with lowerefficiency) transfer the OC to LA-IC. Consequently, thisreduced but still detectable OC production in YeO3-R1-Loswas then the reason for our failure to isolate waaLos

mutants from the transposon insertion library since suchmutants did not survive the enterocoliticin selection.

Analysis of double and triple mutants

To characterize further the functions of WaaLps andWaaLxs we analysed the LPS profiles of the constructeddouble and triple ligase mutants. Results from Westernblotting and silver-stained DOC-PAGE did not reveal aphenotype that could be used to assign a particular func-tion for WaaLxs as the LPS profile of YeO3-c-Lps-Lxs wasidentical to that of YeO3-c-Lps and the wild type (Fig. 2,lanes 1, 5 and 9) and the profile of YeO3-c-Los-Lxs wasidentical to that of YeO3-c-Los (Fig. 2, lanes 4 and 8). Incontrast, in YeO3-c-Los-Lps the elimination of WaaLps

resulted in complete loss of both OC and O-PS (Fig. 2,lane 7). Similarly, the triple mutant YeO3-c-Los-Lps-Lxsproduced neither OC nor O-PS (Fig. 2, lane 10, Table 2).The enterocoliticin sensitivity of the strains was in line withthe OC phenotypes (Table 2).

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To exclude the possibility of additional spontaneousmutations behind the altered LPS phenotypes, plasmidspLos, pLps and pLxs were separately introduced to themutant strains. The OC phenotypes of YeO3-c-Los, YeO3-c-Los-Lps and YeO3-c-Los-Lps-Lxs were complementedin trans by plasmid pLos as indicated by the more inten-sive OC bands in both silver-stained DOC-PAGE andWestern blotting with mAb 2B5, and by enterocoliticin andfR1-37 sensitivities (Fig. 3, lanes 5, 6 and 8, Table 2).However, the complementation was only partial. This isbest illustrated by the presence of strong unsubstituted ICband in the complemented mutants (Fig. 3, silver staining,lanes 5–10) while in the wild-type bacteria the amount of

unsubstituted IC is negligible (Fig. 3, lane 1). Interestingly,pLos also partially complemented O-PS production inYeO3-c-Los/pLos and in YeO3-c-Los-Lps/pLos but not inthe triple mutant (Fig. 3, lanes 5, 6 and 8). On the otherhand, the pLps complemented also the OC and O-PSproduction in strain YeO3-c-Los-Lps/pLps but overall theeffect was smaller than that in YeO3-c-Los-Lps/pLos(Fig. 3, compare lanes 6 and 7). The weak OC-ligaseactivity of pLps is well demonstrated in the complementedtriple mutant (Fig. 3, lane 9). pLxs had no influence on theLPS profile of the triple mutant (Fig. 3, lane 10). Althoughthe restoration of OC and O-PS production was not com-plete by either pLos or pLps, the results indicated that the

Table 1. Bacterial strains, plasmids and bacteriophages used in this work.

Strain Description Reference

Y. enterocolitica6471/76 (= YeO3) Serotype O:3, wild-type strain, patient isolate Skurnik (1984)6471/76-c (= YeO3-c) Virulence plasmid cured derivative of 6471/76 Skurnik (1984)YeO3-R1 Spontaneous rough (O-PS neg) derivative of YeO3-c Al-Hendy et al. (1992)YeO3-c-OC D(wzx-wbcQ), derivative of YeO3-c (OC neg) Biedzka-Sarek et al. (2005)YeO3-c-OC-R Spontaneous rough (O-PS neg) derivative of YeO3-c-OC Biedzka-Sarek et al. (2005)YeO3-c-Los waaLos::pEPlig1727su, ClmR This workYeO3-c-Lps waaLps::pEPlig532su, ClmR This workYeO3-c-Lxs waaLxs::pSW25T-lig777del, SpeR This workYeO3-R1-Los waaLos::pEPlig1727su, ClmR This workYeO3-c-Los-Lps waaLos::pEPlig1727su waaLps::pSW29T-lig532del, ClmR KanR This workYeO3-c-Los-Lxs waaLos::pEPlig1727su waaLxs::pSW25T-lig777del, ClmR SpeR This workYeO3-c-Lps-Lxs waaLps::pEPlig532su waaLxs::pSW25T-lig777del, ClmR SpeR This workYeO3-c-Los-Lps-Lxs waaLos::pEPlig1727su waaLps::pSW29T-lig532del waaLxs::pSW25T-lig777del, ClmR

KanR SpeRThis work

8081-Res Serotype O:8, R-M+, virulence plasmid cured derivative of strain 8081 Zhang and Skurnik (1994)YeO8-c-Los waaLos::pSW23T-lig1727del derivative of 8081-Res, ClmR This work

Escherichia coliHB101/pRK2013 Conjugation helper strain, KanR Ditta et al. (1980)DH5a F-F80lacZDM15 endA recA hsdR(rK

-mK-) supE thi gyrA relA1 D (lacZYA-argF)U169 Woodcock et al. (1989)

S17-1l pir lpir lysogen of S17-1. Host strain for suicide vector constructs and delivery Wilson et al. (1995)w7249 B2163Dnic35, host strain for suicide vector delivery, requirement for diaminopimelic

acid 0.3 mM, KanRBabic et al. (2008)

W3110 rph-1 IN(rrnD-rrnE), wild-type control for waaL mutant M. ValvanoCLM24 DwaaL derivative of W3110 Feldman et al. (2005)

PlasmidspLps waaLps of YeO3 cloned in pTM100, TetR This workpLxs waaLxs of YeO3 cloned in pTM100, TetR This workpLos waaLos of YeO3 cloned in pTM100, TetR This workpEPlig1727su A 602-bp internal fragment of YeO3 waaLos gene cloned in pRV1, ClmR This workpEPlig532su A 544-bp internal fragment of YeO3 waaLps gene cloned in pRV1, ClmR This workpSW23T-lig1727del A 602-bp internal fragment of YeO3 waaLos gene cloned in pSW23T, ClmR This workpSW25T-lig777del A 752-bp internal fragment of YeO3 waaLxs gene cloned in pSW25T, SpeR This workpSW29T-lig532del A 544-bp internal fragment of YeO3 waaLps gene cloned in pSW29T, KanR This workpMF19 wbbLO16 cloned into pEXT21, SpeR Feldman et al. (1999)pSW23T Suicide vector, ClmR Demarre et al. (2005)pSW25T Suicide vector, SpeR Demarre et al. (2005)pSW29T Suicide vector, KanR Demarre et al. (2005)pRV1 Suicide vector, ClmR Skurnik et al. (1995)pTM100 Mobilizable cloning vector, ClmR TetR Michiels and Cornelis (1991)pUC19 Cloning vector, AmpR Yanisch-Perron et al. (1985)

BacteriophagesfYeO3-12 O-PS-specific bacteriophage of Y. enterocolitica O:3 Al-Hendy et al. (1991)fR1-37 OC-specific bacteriophage of Y. enterocolitica O:3 Skurnik et al. (1995)

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LPS phenotypes in the mutants were not caused by spon-taneous mutations.

In summary, the data of these experiments demon-strated that WaaLos, and WaaLps to a lesser extent, contrib-uted on both OC and O-PS ligation, and that the OC- andO-PS-ligase activities were completely abolished only in

the double mutant YeO3-c-Los-Lps (Fig. 2, lane 7). Com-parison of the pLos complementation results of YeO3-c-Los and YeO3-c-Los-Lps indicated a strong OC-ligase rolefor WaaLos (Fig. 3, compare lanes 5 and 6). At the sametime the significantly reduced O-PS production in YeO3-c-Los-Lps/pLos (Fig. 3, lane 6) would indicate that WaaLps

Fig. 2. LPS phenotypes of single, double and triple ligase mutants of Y. enterocolitica O:3 analysed in silver-stained DOC-PAGE and byWestern blotting. The samples were transferred from DOC-PAGE to membrane that was first incubated and developed with the OC-specificmAb 2B5 (middle image) and then without stripping with O-PS-specific mAb TomA6 (right image). The locations of the LPS molecules carryingO-PS, OC or IC are indicated. The strains in different lanes are indicated at the top.

Table 2. Summary of O-PS, OC and ECA phenotypes of single, double and triple ligase mutants of Y. enterocolitica O:3 and their in trans-complemented derivatives analysed by silver-stained DOC-PAGE, Western blotting and by sensitivity to bacteriophages and enterocoliticin.

Strain Genotype Complementation

DOC-PAGE Sensitivity to

Silver Western

fYeO3-12 fR1-37 EnterocoliticinO-PS OC O-PS OC ECA

YeO3-c wt – +++a +++ +++ +++ +++ +++ +++ +++YeO3-R1 Spontaneous rough

mutant– - +++ - +++ +++ - +++ +++

YeO3-c-Los waaLos – + (+) + (+) +++ ++ + -YeO3-c-Los/pLos waaLos WaaLos + ++ ++ ++ +++ ++ ++ +++YeO3-R1-Los waaLos – - - - (+) n.d. - + +YeO3-R1-Los/pLos waaLos WaaLos - ++ - ++ n.d. - ++ +++YeO3-c-Lps waaLps – ++ +++ ++ +++ +++ ++ ++ +++YeO3-c-Lxs waaLxs – +++ +++ +++ +++ +++ ++ ++ +++YeO3-c-Los-Lps waaLos waaLps – - - (+) - +++ - - -YeO3-c-Los-Lps/pLos waaLos waaLps WaaLos - ++ ++ ++ n.d. + ++ ++YeO3-c-Los-Lps/pLps waaLos waaLps WaaLps - - + - n.d. - - -YeO3-c-Los-Lxs waaLos waaLxs – - - + (+) +++ + ++ -YeO3-c-Lps-Lxs waaLps waaLxs – +++ +++ +++ +++ +++ ++ ++ ++YeO3-c-Los-Lps-Lxs waaLos waaLps waaLxs – - - - - +++ - - -YeO3-c-Los-Lps-Lxs/pLos waaLos waaLps waaLxs WaaLos - ++ - ++ n.d - + -YeO3-c-Los-Lps-Lxs/pLps waaLos waaLps waaLxs WaaLps - + - (+) n.d - - -YeO3-c-Los-Lps-Lxs/pLxs waaLos waaLps waaLxs WaaLxs - - - - +++ - - -

a. Relative expression levels compared with wild-type bacteria indicated by (+++). Very weak positive is indicated by (+) and negative by (-). n.d.,not done.

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must play a significant role in O-PS ligation. Future work iswarranted to elucidate these questions.

Role of ligases in ECA expression

The total lack of OC and O-PS was observed in thedouble mutant YeO3-c-Los-Lps indicating that WaaLxs

might not possess any ligase activity for OC or O-PS. Toclarify the ligase specificity of WaaLxs, we tested the con-structed strains for their ECA expression by DOC-PAGEand Western blotting using both proteinase K-treatedwhole-cell lysates and hot phenol/water-extracted LPSsamples. The ECA repeat unit is a trisaccharide that issynthesized on the Und-P carrier lipid analogous to O-unitof heteropolymeric O-PS, and the units are polymerized inthe periplasmic space and then transferred (ligated) eitherto PG or to LPS (Kuhn et al., 1988; Rick et al., 1998;Kajimura et al., 2006). This step also requires ligase activ-ity, and we thought WaaLxs could act as the ECA-specificligase. Western blotting of proteinase K lysates using mAb898 demonstrated that all the single, double and triplemutant strains produced ECAPG similar to the wild-typeYeO3-c control (Table 2) ruling out the role of WaaLxs asECAPG ligase. We also used monovalent rabbit anti-ECAantiserum to detect ECALPS in the mutants (Rabsztynet al., 2011); however, all the strains were positive (datanot shown) indicating that WaaLxs is not the ECALPS ligase.

Distribution of the ligase genes

Table S2 shows the distribution of the closest homologuesof the waaLos, waaLps and waaLxs genes both in genusYersinia and in other bacteria based on TBLASTN searches.Interestingly, it seems that the species can be divided intothree groups: (i) Y. pestis and Y. pseudotuberculosis carryonly the waaLps homologue, (ii) Y. mollaretii, Y. bercovieriand Y. ruckeri carry the waaLps and waaLxs homologues,and (iii) Y. enterocolitica O:8, Y. kristensenii, Y. intermedia,Y. frederiksenii and Y. aldovae carry all three genes.

Role of WaaLos in Y. enterocolitica O:8

Since Y. enterocolitica O:8 expresses a heteropolymericO-PS, but not OC, the role of WaaLos in O:8 was notapparent. To elucidate this we knocked out the waaLos

gene (ye1727) of the O:8 strain 8081-res. Western blot-ting with mAb 1F1 specific for the O:8 O-PS (Gripenberg-Lerche et al., 1994) revealed that wild-type bacteriaexpress a typical population of LPS molecules with c. halfof the molecules carrying a single O-unit. The other halfcarries O-PSs that demonstrate a modal distribution ofchain lengths; preferentially of 6–9 O-units (Fig. 4). InO-antigen polymerase (wzy) mutant all LPS moleculeswould carry a single O-unit (Bengoechea et al., 2002;2004); such LPS is called semi-rough (SR). The SR band

Fig. 3. LPS phenotypes of single waaLos and waaLps mutants and complemented single, double and triple mutants analysed in silver-stainedDOC-PAGE and by Western blotting. The locations of the LPS molecules carrying O-PS, OC or IC are indicated. The samples weretransferred from DOC-PAGE to membrane that was first incubated and developed with the OC-specific mAb 2B5 (middle image) and thenwithout stripping with O-PS-specific mAb TomA6 (right image). The strains in different lanes are indicated at the top.

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was almost completely missing from the YeO8-c-Los LPSindicating that the role of WaaLos in O:8 was similar to itshomologue in O:3; to ligate an OS to LA-IC thus justifyingthe use of the more general name WaaLos for this ligase.Apparently WaaLos in O:8 ensures that half of the LPSmolecules are substituted with a single O-unit. The bio-logical role of this phenomenon remains to be elucidated.

The OC and O-PS substitutions exclude each other insingle LPS molecule of Y. enterocolitica O:3

The specificity of a ligase is likely to be based on thedifferences between the ligated saccharide moieties,between the target structures, or on both. The ligase couldbe specific for the size of the transferred saccharide (OSversus PS) or for the reducing end sugar; in Y. entero-colitica O:3 the latter is Sugp (2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose) in OC (Zhang, 1996; Skurnik,1999; Pinta et al., 2009) and GlcNAc (2-acetamido-2-deoxy-D-glucopyranose) in O-PS (Zhang, 1996; Skurnik,1999; Pinta et al., 2009). While Sugp of OC is known to beb(1-3) linked to the L,D-heptose II (Hep II) of the IC (Pintaet al., 2009) the linkage from the GlcNAc of O-PS to IC isstill unknown. It has been suggested that O-PS could belinked to one of the Glc (D-glucopyranose) residues of theIC and if this was the case OC and O-PS might substitutea single LPS molecule. On the other hand, the WaaLps

homologue is the only ligase that is present in Y. pestis andit transfers a single GlcNAc residue to Hep II (Anisimovet al., 2010). In addition, the Y. pseudotuberculosis WaaLps

homologue likely ligates O-PS to Hep II (Knirel et al., 2008)and, furthermore, both the Y. enterocolitica O:3 OC and

O-PS can be ligated by Y. pestis WaaLps to its LPS (Kil-junen et al., 2005; 2011). Finally, the fact that WaaLos canligate both OC and O-PS (Fig. 3, lane 2) suggests that theboth OC and O-PS are ligated to Hep II. Furthermore, theWaaL proteins appear to require a specific LA-IC acceptorstructure for ligase activity and from the donor they ratherrecognize the Und-P carrier than the saccharide attachedto it (Heinrichs et al., 1998; Guo et al., 2008). Takentogether, it would be likely that both WaaLos and WaaLps

target to Hep II. As a consequence, OC and O-PS wouldnot co-substitute a single LPS molecule.

The above reasoning would contradict our earlierproposition that in Y. enterocolitica O:3 both O-PS and OCwould reside in a single LPS molecule where the OCwould form a branch (Skurnik et al., 1995; 1999). Thisearlier scenario was further opposed by the Westernblotting data presented in Fig. 5. The O-PS-specific mAbTomA6 reacted only with the homopolymeric O-PS smearof YeO3-c proteinase K lysates in the upper part of the gel.In the same sample, the OC-specific mAb 2B5 reactedwith the sharp LA-IC-OC band in the lower part of the gel(Fig. 5). If both OC and O-PS were linked to the sameLPS molecule, OC should also be detected from theregion stained by the O-PS-specific mAb. However, wehave never seen mAb 2B5 reacting with the O-PS-

Fig. 4. Role of WaaLos in Y. enterocolitica O:8. Western blottingwith O-PS-specific mAb 1F1. The locations of the semi-rough (SR)band and the O-PS bands are indicated. Note the significantlydecreased intensity of the SR band in YeO8-c-Los.

Fig. 5. The OC and O-PS of Y. enterocolitica O:3 reside ondifferent LPS molecules. Western blotting analysis of LPS samplesof YeO3-c. Strain YeO3-c-OC-R was used as OC- andO-PS-negative control. The locations of the LPS molecules carryingO-PS or OC are indicated. In the left image pair, the membranestrip with blotted YeO3-c sample was first incubated and developedwith the OC-specific mAb 2B5 and then without stripping withO-PS-specific mAb TomA6. In the right image pair are duplicatemembranes; the left-side membrane was developed with mAb 2B5and the right-side membrane with mAb TomA6.

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containing smear (Fig. 5, see also Figs 2 and 3) even withextended exposure (data not shown) suggesting thatthere is no OC in those molecules. The possibility that the2B5 epitope would be hidden by O-PS is highly unlikelyespecially as the LPS molecules were separated in theDOC-PAGE. We therefore suggest that in Y. enterocoliticaO:3 a single LPS molecule only carries either OC or O-PSlinked to LA-IC. Furthermore, we predict that also O-PS islinked to Hep II with a b(1-3) linkage although directchemical evidence for the linkage is still missing. Weenvision that a single wild-type Y. enterocolitica O:3bacterium carries a mixture of LPS molecules on itssurface; approximately equal numbers of OC- and O-PS-substituted and a few naked LA-IC molecules (Fig. 2, lane1). It should be noted that in the O-PS-lacking strainYeO3-R1 all the LPS molecules were substituted by OC(Fig. 2, lane 3); however, in the OC-lacking strain YeO3-c-OC the proportion of the O-PS-substituted moleculeswas almost the same as in the wild-type bacteria, and asignificant amount of the LPS molecules remained asnaked LA-IC molecules (Fig. 2, lane 2). Thus, evenwithout competition from OC ligation the bacteria are ableto control that O-PS is ligated only to about half of theavailable LA-IC molecules. Such selectivity could be dueto structural heterogeneity of the LA-IC molecules; onlyhalf of them would be able to accept O-PS, or to the LPStransport pathway that translocates the LPS molecules toOM (Ruiz et al., 2009). It will be interesting to elucidatehow bacteria control the balance of OC and O-PS substi-tutions on the LPS.

Conclusions

In this work we identified from Y. enterocolitica O:3 twoligases involved in the biosynthesis of LPS and a thirdligase with unknown function. Due to promiscuity of theligases, our strategy to identify the OC ligase by screen-ing a transposon insertion library for OC-non-expressingmutants was not successful and in the end we identifiedthe ligase genes using bioinformatics. The predictedamino acid sequences of the ligases were c. 50% iden-tical and demonstrated a ligase-characteristic membranetopology pattern. All the ligases demonstrated O-PS-ligase activity in E. coli DwaaL strain; however, charac-terization of single, double and triple ligase mutantsindicated that in Y. enterocolitica O:3 only two of theligases are LPS ligases. The function of WaaLxs remainedunresolved. The predicted C-terminal c. 100-amino-acid-long cytoplasmic domain in WaaLxs resembles that ofoligosaccharyltransferases such as PglL of Neisseriameningitidis involved in bacterial protein O-glycosylation(Faridmoayer et al., 2008). These transferases are highlypromiscuous that would explain the O-PS-ligase activityof Waalxs in E. coli DwaaL strain. The characterization of

the role of WaaLxs in protein glycosylation of Y. entero-colitica O:3, if any, needs further studies.

To our knowledge this is the first time that more than oneligase has been experimentally proven to be involved in theLPS biosynthesis of a single bacterial strain. In the otherorganisms studied this far, such as E. coli and Salmonellaonly one waaL gene has been characterized to be respon-sible for the ligation reactions concerning LPS biosynthe-sis. Interestingly, Y. pestis and Y. pseudotuberculosis haveonly the WaaLps homologue, while one or both of the otherligases are additionally present in other Yersinia species.The rationale for having two or three different ligases in asingle bacterium is not directly apparent. Further work isrequired to elucidate the biological significance of thesedifferent settings.

Experimental procedures

Bacterial strains, plasmids, bacteriophages and cultureconditions

Bacterial strains, plasmids and bacteriophages used in thiswork are listed in Table 1. Yersinia strains were grown intryptic soy broth (TSB) at 20–22°C (RT) unless otherwisementioned and E. coli strains in lysogeny broth (LB) (Bertani,2004) at 37°C. LB supplemented with 1.5% Bacto Agar wasused for all solid cultures. When required, appropriate anti-biotics were added as follows: chloramphenicol (Clm)20 mg ml-1, ampicillin (Amp) 100 mg ml-1, tetracycline (Tet)12.5 mg ml-1, kanamycin (Kan) 100 mg ml-1, spectinomycin(Spe) 80 mg ml-1 and diaminopimelic acid (Dap) 0.3 mM.

Construction and screening of the transposon insertionlibrary of YeO3-R1

Details of the in vitro transposon insertion protocol andscreening of enterocoliticin-resistant strains are described inSupporting information.

General DNA techniques

Isolations of plasmid and genomic DNA as well as Southernanalysis were done as previously described (Sambrook andRussell, 2001). PCR analysis of transposon insertion mutantswas made with the Expand Long PCR Polymerase System(Roche) according to manufacturer’s suggestions withprimers Yerfb5, Yerfb6, Muc1 and Muc2 (Table S1). GenomicDNA isolated from the mutants was used as a template.

Construction of mutants

The 602-, 752- and 544-bp internal fragments of the waaLos,waaLxs and waaLps genes (nucleotides 156–758, 777–1529and 221–775 of the respective open reading frames) wereamplified by PCR with primer pairs 1727delF & 1727delR,777delF & 777delR and 532delF & 532delR (Table S1),respectively, and Phusion DNA polymerase (Finnzymes)

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according to supplier’s recommendations. The gel-purifiedand XhoI-digested waaLos and waaLps fragments were clonedinto SalI-digested suicide vectors pRV1, pSW23T andpSW29T to obtain plasmids pEPlig1727su, pSW23T-lig1727del, pEPlig532su and pSW29T-lig532del (Table 1).The purified waaLxs fragment was phosphorylated by T4 poly-nucleotide kinase in the presence of ATP, gel-purified andligated with SmaI-digested, shrimp alkaline phosphatase(SAP)-treated and gel-purified pSW25T. The obtained plasmidwas named pSW25T-lig777del.

The constructed suicide vectors were mobilized to appro-priate recipient strains by conjugation as described earlier(Biedzka-Sarek et al., 2005) and the resulting single, doubleand triple mutants are listed in Table 1. The integration of thesuicide plasmids to the host genome by homologous recom-bination between the cloned gene fragments and the targetgenes interrupted the target genes. The genotypes of themutants were confirmed by Southern blotting analysis withDIG-labelled gene-specific probes (Roche) and by PCR withprimers that amplify the full-length genes (Table S1). Themutant strains gave PCR products of about 5 kb (correspond-ing to the gene, internal fragment and the vector all together).DNA of wild-type strain YeO3-c was used as a control.

Complementation of the Y. enterocolitica O:3ligase mutants

The full ORFs of waaLos, waaLps and waaLxs genes of YeO3-cwere cloned as 1.2-, 1.2- and 1.7-kb PCR fragments [ampli-fied with Phusion DNA polymerase using primer pairsO3ligYe1727f & O3ligYe1727r, O3ligYe532f & O3ligYe532rand O3ligYe777f & O3ligYe777r (Table S1) respectively] intoplasmid pTM100 to obtain plasmids pLos, pLps and pLxs(Table 1). Briefly, the PCR fragments were phosphorylatedwith polynucleotide kinase (Fermentas) in the presence ofATP, digested with EcoRI and ligated with EcoRI&ScaI-digested, SAP-treated pTM100. The constructed plasmidswere mobilized to the different ligase mutants by triparentalconjugation as described earlier (Biedzka-Sarek et al., 2005).

Complementation of the E. coli DwaaL mutant

The complementation experiments were performed asdescribed earlier (Perez et al., 2008). Briefly, the plasmidspLos, pLxs and pLps were introduced by electroporation toE. coli DwaaL strain CLM24 together with plasmid pMF19, andthe constructed strains were named CLM24/pMF19,pLos,CLM24/pMF19,pLxs and CLM24/pMF19,pLps respectively.The plasmid pMF19 was also introduced to strains CLM24 andW3110 and the strains were named CLM24/pMF19 andW3110/pMF19 respectively.

DOC-PAGE analysis and Western blotting

The bacteria were grown for 16–20 h at RT in 5 ml of TSBmedium with appropriate antibiotics. The exact optical densityat 600 nm (OD600) of the cultures was measured, 1 ml of thecultures were centrifuged and the pellets were resuspended indeoxycholate lysis buffer (2% DOC, 4% 2-mercaptoethanol,

10% glycerol and 0.002% bromophenol blue in 1 M Tris-HClbuffer, pH 6.8) in a volume adjusted according to density of theculture (100 ml/OD600 = 1). LPS phenotypes of proteinaseK-treated whole cell lysates (for ECA analysis also hot phenol/water-extracted samples) were analysed by silver-stainedDOC-PAGE (Zhang and Skurnik, 1994; Skurnik et al., 1995)and by Western blotting as described earlier (Biedzka-Sareket al., 2005).

Antibodies and antisera

Murine mAb 2B5 and TomA6 are specific for Y. enterocoliticaO:3 OC and O-PS, respectively (Pekkola-Heino et al., 1987;Pinta et al., 2010), and mAb 898 is specific for ECAPG (Peterset al., 1985). Monovalent rabbit anti-ECA antiserum is spe-cific for ECALPS of Y. enterocolitica O:3 (Rabsztyn et al.,2011). mAb 1F1 is specific for the O:8 O-PS (Gripenberg-Lerche et al., 1994).

fR1-37, fYeO3-12 and enterocoliticin sensitivity assays

Different strains were tested for their fR1-37, fYeO3-12 andenterocoliticin sensitivities by pipetting 5 ml of serial dilutionsof phage or enterocoliticin stocks (2 ¥ 109 pfu ml-1 and 2 ¥ 106

activity units ml-1 respectively) on a freshly grown and driedbacterial lawn and observing the formation of a clear lysiszone after 24 h incubation at RT.

Acknowledgements

We thank Jukka Karhu for excellent technical assistance, DrEckhard Strauch for providing the enterocoliticin and DrXiang Ruan and Dr Miguel Valvano for the help with E. coliDwaaL complementation experiments (Fig. S2). This workwas supported by Academy of Finland (projects 114075,104361 and 50441) to M.S. and Finnish GlycoscienceGraduate School to E.P.

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