Molecular and Functional Characterization of O Antigen Transfer inVibriocholerae
2005; Elsevier BV; Volume: 280; Issue: 27 Linguagem: Inglês
10.1074/jbc.m501259200
ISSN1083-351X
AutoresStefan Schild, Anna‐Karina Lamprecht, Joachim Reidl,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe majority of Gram-negative bacteria transfer O antigen polysaccharidesonto the lipid A-core oligosaccharide via the action of surface polymer:lipidA-core ligases (WaaL). Here, we characterize the WaaL proteins of Vibriocholerae with emphasis on structural and functional characterization of Oantigen transfer and core oligosaccharide recognition. We demonstrate that theactivity of two distantly related O antigen ligases is dependent on thepresence of N-acetylglucosamine, and substitution of an additionalsugar, i.e. galactose, alters the site specificity of the coreoligosaccharide necessitating discriminative WaaL types. Protein topologyanalysis and a conserved domain search identified two distinct conservedmotifs in the periplasmic domains of WaaL proteins. Site-directed mutagenesisof the two motifs, shown for WaaLs of V. cholerae and Salmonellaenterica, caused a loss of O antigen transfer activity. Moreover, analogyof topology and motifs between WaaLs and O polysaccharide polymerases (Wzy)reveals a relationship between the two protein families, suggesting that thecatalyzed reactions are related to each other. The majority of Gram-negative bacteria transfer O antigen polysaccharidesonto the lipid A-core oligosaccharide via the action of surface polymer:lipidA-core ligases (WaaL). Here, we characterize the WaaL proteins of Vibriocholerae with emphasis on structural and functional characterization of Oantigen transfer and core oligosaccharide recognition. We demonstrate that theactivity of two distantly related O antigen ligases is dependent on thepresence of N-acetylglucosamine, and substitution of an additionalsugar, i.e. galactose, alters the site specificity of the coreoligosaccharide necessitating discriminative WaaL types. Protein topologyanalysis and a conserved domain search identified two distinct conservedmotifs in the periplasmic domains of WaaL proteins. Site-directed mutagenesisof the two motifs, shown for WaaLs of V. cholerae and Salmonellaenterica, caused a loss of O antigen transfer activity. Moreover, analogyof topology and motifs between WaaLs and O polysaccharide polymerases (Wzy)reveals a relationship between the two protein families, suggesting that thecatalyzed reactions are related to each other. The outer lipid leaflet of the outer membrane of Gram-negative bacteria(1Kamio Y. Nikaido H. Biochemistry. 1976; 15: 2561-2570Crossref PubMed Scopus (234) Google Scholar) is composed almostexclusively of lipopolysaccharide(LPS) 1The abbreviations used are: LPS, lipopolysaccharide; ampr,ampicillin-resistant; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; core OS,core oligosaccharide; kanr, kanamycin-resistant; LB, Luria broth;strepr, streptomycin-resistant; und-PP, undecaprenyl pyrophosphate;X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoyranoside.1The abbreviations used are: LPS, lipopolysaccharide; ampr,ampicillin-resistant; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; core OS,core oligosaccharide; kanr, kanamycin-resistant; LB, Luria broth;strepr, streptomycin-resistant; und-PP, undecaprenyl pyrophosphate;X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoyranoside. consisting ofthree complex portions, i.e. the lipid A, the core oligosaccharides(core OS), and the O polysaccharides. As a polyanionic lipid, the LPS leafletacts as an effective barrier for the diffusion of lipophilic and hydrophobiccompounds. This characteristic depends on the lipid A-core OS complex (for arecent summary, see Ref. 2Nikaido H. Microb. Mol. Biol.Rev. 2003; 67: 593-656Crossref PubMed Scopus (2714) Google Scholar). Inpathogenic bacteria O polysaccharides serve important biological functions indisease. They protect against the host immune recognition system, complementattack, the immune response, are involved in mediating adherence to hostsurfaces, and they produce host mimicry(2Nikaido H. Microb. Mol. Biol.Rev. 2003; 67: 593-656Crossref PubMed Scopus (2714) Google Scholar, 3Harvey H.A. Swords W.E. Apicella M.A. J. Autoimmun. 2001; 16: 257-262Crossref PubMed Scopus (61) Google Scholar, 4Moran A.P. J. Physiol.Pharmacol. 1999; 50: 787-805PubMed Google Scholar, 5Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar, 6Paton A.W. Voss E. 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Microbiol. 1998; 20: 45-54Crossref PubMed Scopus (29) Google Scholar).The O polysaccharide structures of the cholera causing serogroups O1 and O139(13Kenne L. Lindberg B. Unger P. Gustafsson B. Holme T. Carbohydr. Res. 1982; 100: 341-349Crossref PubMed Scopus (113) Google Scholar,14Knirel Y.A. Widmalm G. Senchenkova S.N. Jansson P-E. andWeintraub A. Eur. J. Biochem. 1997; 247: 402-410Crossref PubMed Scopus (46) Google Scholar), as well as the underlyingcore OS, have been resolved(15Vinogradov E.V. Bock K. Holst O. Brade H. Eur. J. Biochem. 1995; 233: 152-158Crossref PubMed Scopus (40) Google Scholar,16Cox A.D. Perry M.B. Carbohydr. Res. 1996; 290: 59-65Crossref PubMed Scopus (49) Google Scholar). Compared with O1, thecore OS of two non-O1 isolates, H11(17Bock K. Vinegradov E.V. Holst O. Brade H. Eur. J. Biochem. 1994; 225: 1029-1039Crossref PubMed Scopus (54) Google Scholar), and O22(14Knirel Y.A. Widmalm G. Senchenkova S.N. Jansson P-E. andWeintraub A. Eur. J. Biochem. 1997; 247: 402-410Crossref PubMed Scopus (46) Google Scholar,18Cox A.D. Brisson J.R. Thibault P. Perry M.B. Carbohydr. Res. 1997; 304: 191-208Crossref PubMed Scopus (20) Google Scholar), revealed distinctstructural differences. In addition, genetic analysis of the wavgenes (for core OS biosynthesis) of various environmental isolates of V.cholerae shows distinct genetic variation(19Nesper J. Kraiss A. Schild S. Blass J. Klose K.E. Bockemühl J. Reidl J. Infect.Immun. 2002; 70: 2419-2433Crossref PubMed Scopus (41) Google Scholar). Thus, core OSvariability does exist within V. cholerae as found in Escherichiacoli (20Amor K. Heinrichs D. Frirdich E. Ziebell K. Johnson R. Whitfield C. Infect. Immun. 2000; 68: 1116-1124Crossref PubMed Scopus (132) Google Scholar). Many aspects of LPS biosynthesis have been resolved, and it is clear thatlipid A serves as an acceptor for core OS synthesis. Core OS synthesis iscoordinately achieved by the interplay of highly specific heptosyl- andglycosyltransferases, which are associated with the inner side of thecytoplasmic membrane. After completion of synthesis, the lipid A-core OSmolecules are subsequently transported to the periplasmic compartment via MsbA(for recent review, see Ref.5Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). For the O polysaccharides,three different synthesis and transport systems are known, which are all basedon the transfer of capped und-PP-linked O polysaccharides, delivered to theperiplasm either by the Wzy, ABC transporters, or synthase-dependent pathways(5Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). The subsequent ligation ofO polysaccharide and lipid A-core OS takes place in the periplasm(21Mulford C.A. Osborn M.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1159-1163Crossref PubMed Scopus (70) Google Scholar). The key proteinresponsible for this reaction was identified as a surface polymer:lipid A-coreligase (O antigen ligase), encoded by the waaL gene as summarizedrecently (5Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar,22Yethon J.A. Whitfield C. Curr. Drug Targets Infect. Disord. 2001; 1: 91-106Crossref PubMed Scopus (71) Google Scholar). Based on complementationstudies in Enterobacteriaceae, it was suggested that the O antigen ligases(23MacLachlan P.R. Kadam S.K. Sanderson K.E. J. Bacteriol. 1991; 173: 7151-7163Crossref PubMed Google Scholar,24Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar) are membrane-associatedand seem to possess substrate specificity for lipid A-core OS, but they arerelaxed in their recognition of the O polysaccharides(25Whitfield C. Amor P.A. Köplin R. Mol. Microbiol. 1997; 23: 629-638Crossref PubMed Scopus (125) Google Scholar,26Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 8849-8859Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, since the firstgenetic evidence for WaaL function(27Schmidt G. Jann B. Jann K. Eur. J. Biochem. 1970; 16: 382-392Crossref PubMed Scopus (47) Google Scholar), no direct biochemicaldata have been produced which prove that the WaaL proteins are enzymes.Therefore, the important final step in the synthesis of the LPS molecule isstill not fully understood. Using WaaL of V. cholerae andSalmonella enterica sv. Typhimurium, we investigated coreOS-dependent WaaL specificity, ligase activity, and WaaL membranetopology. Bacterial Strains, Plasmids, and Media—Except as notedotherwise, for all genetic manipulations E. coli strain XL1-blue (NewEngland Biolabs, Inc.), Sm10λpir(28Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1699) Google Scholar), and CC118(29Manoil C. Beckwith J. Proc.Natl. Acad. Sci. U. S. A. 1985; 82: 8129-8133Crossref PubMed Scopus (655) Google Scholar) were used; otherbacterial strains and plasmids used are listed inTable I. Bacteria were grown inLuria broth (LB, BD Biosciences) at 37 °C with aeration. Antibiotics(Sigma) and other medium supplements were used in the followingconcentrations: 100 μg/ml streptomycin, 50 μg/ml kanamycin, and 50 or100 μg/ml ampicillin for V. cholerae or E. coli,respectively. For TnphoA, TnlacZ mutagenesis in E.coli kanamycin was used at up to 300 μg/ml. For determining PhoA andLacZ activity 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) (40μg/ml) were used, respectively. V. cholerae strain O1 and O139,referred to in this manuscript, are the O1 El Tor strain P27459 and the O139strain MO10, respectively (see TableI).Table IBacteria strains used in this studyStrainRelevant genotype and/or phenotypeSourceE. coli and S. entericaBL21 (DE3)F-ompT hsdSB(rB-mB-) gal dcm (DE3)InvitrogenBL21 (DE3) pLysSF-ompT hsdSB(rB-mB-)Invitrogengal dcm (DE3) pLysS (cmr)CC313CC118 pOxygen (TnlacZ)C. ManoilIS212KS272 λ11.10, bor::TnphoA(37Barondess J. Beckwith J. Nature. 1990; 346: 871-874Crossref PubMed Scopus (139) Google Scholar)TOP10FF′ (laqq,Tn10(TcR))mcrAΔ(mrr-hsdRMS-mcrBC)φ80lacZΔM15ΔlacX74 recA1 deoRaraD139Δ(ara-leu)7697 galU galK rpsL (Smr)endA1 nupGInvitrogenSARC6S. enterica sv. ArizonaeIIIA (serotype 62:z36:-)(51Boyd E.F. Wang F.S. Whittam T.S. Selander R.K. Appl. Environ. Microbiol. 1996; 62: 804-808Crossref PubMed Google Scholar)CWG620SARC6 waaL, cmr(51Boyd E.F. Wang F.S. Whittam T.S. Selander R.K. Appl. Environ. Microbiol. 1996; 62: 804-808Crossref PubMed Google Scholar)Sm10λpirthi thr leu tonA lacY supE recA::RP4-2-Tc::MuλpirRK6, kanr(28Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1699) Google Scholar)XL1-blueF′::Tn10 proA + B +lacqΔ(lacZ)M15/recA1 endA1gyrA46 (Nalr) thi hsdR17(rK- mK+) supE44 relA1lacNew England BiolabsV. choleraeO1O1 El Tor P27459, strepr(32Nesper J. Kapfhammer D. Klose K.E. Merkert H. Reidl J. J. Bacteriol. 2000; 182: 5097-5104Crossref PubMed Scopus (49) Google Scholar)O1waaLP27459waaL::pGP in P27459-S, ampr(19Nesper J. Kraiss A. Schild S. Blass J. Klose K.E. Bockemühl J. Reidl J. Infect.Immun. 2002; 70: 2419-2433Crossref PubMed Scopus (41) Google Scholar)O1ΔwavLP27459, ΔwavL(Δaa177-289)This studyO1Δ1wavLP27459, ΔwavL(Δaa177-483)This studyO1wavKwavK::pGP in P27459, amprThis studyO1wavIwavI::pGP in P27459, amprThis studyO1wavGwavG::pGP in P27459, amprThis studyO1wavFwavF::pGP in P27459, amprThis studyO1wavEwavE::pGP in P27459, amprThis studyO1wavAwavA::pGP in P27459, amprThis studyO1wavAΔwavLwavA::pGP in P27459ΔwavL, amprThis studyV194Environmental isolate(19Nesper J. Kraiss A. Schild S. Blass J. Klose K.E. Bockemühl J. Reidl J. Infect.Immun. 2002; 70: 2419-2433Crossref PubMed Scopus (41) Google Scholar)SV194V194, spontaneous streprThis studySV194waaLwaaL::pGP in SV194, amprThis studySV194wavMwavM::pGP in SV194, amprThis studySV194wavLwavL::pGP in SV194, amprThis studyO139O139, strepr, cmr(11Waldor M.K. Colwell R. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11388-11392Crossref PubMed Scopus (166) Google Scholar)O139wavLwavL::pGP in MO10, amprThis studyO139ΔwaaLΔwaaL(42Nesper J. Schild S. Lauriano C.M. Kraiss A. Klose K.E. Reidl J. Infect. Immun. 2002; 70: 5990-5996Crossref PubMed Scopus (50) Google Scholar) Open table in a new tab PCR, DNA Purification, DNA Sequencing, and Southern BlotAnalysis—PCR products or digested plasmid DNA was purified usingthe Qiaquick Gel Extraction or Qiaquick PCR Purification Kit (Qiagen). PCRsfor sequencing and subcloning were carried out using the Triple Master system(Eppendorf). Automated DNA sequencing was performed with an ABI 377 using thedye terminator cycle method with AmpliTaq (Applied Biosystems). ChromosomalDNA was prepared as described by Grimberg et al.(30Grimberg J. Maguire S. Belluscio L. Nucleic Acids Res. 1989; 17: 8893Crossref PubMed Scopus (66) Google Scholar). Southern blot analysiswas performed as described by Southern(31Southern E.M. J. Mol.Biol. 1975; 51: 503-517Crossref Scopus (21353) Google Scholar). Construction of Suicide Plasmids—All insertion mutants usingthe suicide plasmid pGP704 were constructed in a similar manner. An internalfragment of the respective gene was amplified by PCR using oligonucleotideswith SacI or XbaI restriction sites, as listed inTable II. For the constructionof pGPwaaLV194, pGPwavMV194 and pGPwavLV194 chromosomal DNA of strain V194served as template. In all other cases chromosomal DNA of strain O1 wasutilized. SacI/XbaI-digested PCR fragments were ligated into pGP704 digestedwith SacI/XbaI and transformed into Sm10λpir. Ampr colonieswere characterized by restriction analysis and PCR.Table IIPlasmids used in this studyPlasmidRelevant genotype/resistanceSourcepCRT7/ CT-TOPOamprInvitrogenpGP704oriR6K mobRP4, ampr(28Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1699) Google Scholar)pKEK229oriR6K mobRP4 sacB, ampr(52Correa N.E. Lauriano C.M. McGee R. Klose K.E. Mol. Microbiol. 2000; 35: 743-755Crossref PubMed Scopus (87) Google Scholar)pOxygenTnlacZ, kanr(38Manoil C. Methods CellBiol. 1991; 34: 61-75Crossref PubMed Scopus (196) Google Scholar)pBAD18-Kankanr(53Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar)pBAD18ampr(53Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar)pTrc99Aampr(54Amann E. Ochs B. Abel K.J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (868) Google Scholar)pWQ322ampr, waaL of SARC6C. WhitfieldpGEM-T easyamprPromegapGPwavLwavL of P27459 in pGP704This studypGPwavKwavK of P27459 in pGP704This studypGPwavIwaaI of P27459 in pGP704This studypGPwavGwavG of P27459 in pGP704This studypGPwavFwavF of P27459 in pGP704This studypGPwavEwavE of P27459 in pGP704This studypGPwavAwavA of P27459 in pGP704This studypGPwaaLV194waaL of V194 in pGP704This studypGPwavMV194wavM of V194 in pGP704This studypGPwavLV194wavL of V194 in pGP704This studypKEKΔwavLΔwavL of P27459 in pKEK229This studypKEKΔ1wavLΔ1wavL of P27459 in pKEK229This studypBAD18-KanwaaLO1waaL of P27459 in pBAD18-Kan(19Nesper J. Kraiss A. Schild S. Blass J. Klose K.E. Bockemühl J. Reidl J. Infect.Immun. 2002; 70: 2419-2433Crossref PubMed Scopus (41) Google Scholar)pwaaLO1waaL of P27459 subcloned from pBAD-KanwaaL in pBAD18This studypwaaLV194waaL of V194 in pBAD18-KanThis studypwavMV194wavM of V194 in pBAD18-KanThis studypwaaLwavMV194wavM and waaL of V194 in pBAD18-KanThis studypTrcwavLwavL of P27459 in pTrc99AThis studyphybrid-waaLO1/V194N-terminal part of waaL of P27459 and C-terminal part ofwaaL of V194 linked by NcoI in pBAD18-KanThis studyphybrid-waaLV194/O1N-terminal part of waaL of V194 and C-terminal part of waaLof P27459 linked by NcoI in pBAD18-KanThis studypwaaLO1::TnlacZTnlacZ hybridfusion plasmids, kanr, amprThis studypwaaLO1::TnphoATnphoA hybrid fusion plasmids, kanr, amprThis studypQE30amprQiagenpQE30waaLO1waaL of P27459 in pQE30This studypQE30waaLO1-H311AwaaL H311A of P27459 in pQE30This studypQE30waaLO1-R186MwaaL R186M of P27459 in pQE30This studypQE30waaLO1-H309AwaaL H309A of P27459 in pQE30This studypQE30waaLO1-H309A/H311AwaaL H309A/H311A of P27459 in pQE30This studypTOPOwavLwavL of P27459 in pCRT7/CT-TOPOThis studypTOPOwavMwavM of V194 in pCRT7/CT-TOPOThis studypGEMwavLwavL of P27459 in pGEM-T easyThis studypGEMwavLNN-terminal wavL fragment (amino acids 1- 361) of P27459 in pGEM-TeasyThis studypWQ322-H321AwaaL H321A in pWQ322This studypWQ322-R208MwaaL R208M in pWQ322This study Open table in a new tab The suicide plasmids pKEKΔwavL and pKEKΔ1wavL were constructedto introduce a 336-bp deletion for ΔwavL and 918 bp forΔ1wavL by generating two PCR fragments using theoligonucleotide pairs wavL-SacI-intern5′ wavL-BamHI-intern5′, andwavL-BamHI-intern3′, wavL-XbaI-intern3′ pKEKΔwavL.pKEKΔ1wavL was constructed using the oligonucleotide pairswavL-SacI-intern5′ wavL-BamHI-intern5′ andwavL-BamHI-intern1–3′ wavL-XbaI-intern3′. PCR fragments weretreated with BamHI and ligated together. The ligation product was amplified byPCR using oligonucleotides wavL-SacI-intern5′ andwavL-XbaI-intern3′, subsequently digested with SacI/XbaI, and ligated ina SacI/XbaI-digested pKEK229. After transformation of the ligation mix intoSm10λpir, ampr colonies were characterized by restrictionanalysis and PCR (data not shown). Construction of Mutant Strains—The suicide plasmids wereconjugated into V. cholerae O1, O139, or SV194, and integration ontothe chromosome was selected by isolation of ampr/streprcolonies, as described before(32Nesper J. Kapfhammer D. Klose K.E. Merkert H. Reidl J. J. Bacteriol. 2000; 182: 5097-5104Crossref PubMed Scopus (49) Google Scholar). For the construction ofthe deletion mutants O1ΔwavL and O1Δ1wavLsucrose selection was used to obtain amps colonies. The correctinsertions or chromosomal deletions of the constructs were confirmed by PCRand Southern blot analysis (data not shown). Construction of Expression Plasmids—For the construction ofthe plasmids pTrcwavL and pGEMwavL, the wavL gene was amplified byPCR using the oligonucleotides wavL-5′-EcoRI and wavL-3′-BamHI andchromosomal DNA of O1 as template. The purified PCR product was either ligateddirectly into pGEM-T easy (Promega) or digested with EcoRI/BamHI and ligatedinto an EcoRI/BamHI-digested pTrc99A. To construct pGEMwavLN theN-terminal part of the gene was amplified by PCR using the oligonucleotideswavL-5′-EcoRI and wavL-N-term-up and chromosomal DNA of O1 as template,then the purified PCR product was ligated into pGEM-T easy. The expression plasmids pwaaLV194 and pwaaLwavMV194were constructed by amplifying the genes using oligonucleotide pairswaaLV194-5′-SacI, waaL-V194-3′-KpnI, and wavM-V194-5′-SacI,waaLV194-3′-KpnI, using chromosomal DNA of V194 as template. The PCRproducts were treated with SacI/KpnI, then ligated into SacI/KpnI-digestedpBAD18-Kan. Digestion of pwaaLwavMV194 with XbaI and religationresulted in the plasmid pwavMV194 lacking waaL of V194.After transformation of the ligation products into XL1-blue, amprcolonies for pTrcwavL, pGEMwavL, and pGEMwavLN or kanrcolonies (in the other cases) were characterized by restriction analysis andPCR (data not shown). Complementation was observed by supplementing the mediawith 1 mm isopropyl-β-d-thiogalactopyranoside, inthe case of pTrcwavL, pGEMwavL, and pGEMwavLN, and 0.02% arabinosefor pwaaLV194, pwaaLwavMV194, andpwavMV194. To analyze the O antigen ligase activity, the hybrid plasmids ofwaaL, phybrid-waaLO1/V194, andphybrid-waaLV194/O1, consisting of N- and C-terminal parts ofwaaL derived from V194 and O1, were constructed. N-terminal portions(N-cassettes) of waaL were amplified by PCR using oligonucleotidepairs waaL-O1-5′-NheI and waaL-O1-up-NcoI in the case of O1, andoligonucleotide pairs waaL-V194-5′-NheI and waaL-V194-up-NcoI in thecase of V194. C-terminal parts (C-cassettes) of waaL were amplifiedby PCR using oligonucleotide pairs waaL-O1-3′-KpnI and waaL-O1-down-NcoIin the case of O1, and oligonucleotide pairs waaL-V194-3′-KpnI andwaaL-V194-down-NcoI in the case of V194. PCR products containing theN-terminal portions of waaL were treated with NcoI/NheI and ligatedto C-terminal portions of waaL that had been digested with NcoI/KpnI,along with an NheI/KpnI-digested pBAD18-Kan in a three-body ligation reaction.The fusion construct of hybrid WaaLV194-WaaLO1 consistsof the N-terminal 220 amino acids of WaaLV194 fused to 186 aminoacids of the C-terminal portion of waaLO1 ligase, startingat position Val214. The connecting region of this construct alsocontains an additional Ala and Met. The other fusion construct of hybridWaaLO1-WaaLV194 consists of the N-terminal 212 aminoacids of WaaLO1 fused to 180 amino acids of the C-terminal portionof V194 ligase, starting at position Thr224. The connecting regionof this construct harbors an additional Met and Val. After transformation ofthe ligation products into XL1-blue, kanr colonies werecharacterized by restriction analysis and PCR (data not shown). Byconstructing these hybrids between N- and C-cassettes of waaLs wealso reconstructed waaLO1 and waaLV194with the NcoI restriction site and subsequently showed that they were activein O antigen ligase complementation in the respective waaL mutants(data not shown). Construction of His-tagged WavL and WavM Expression Systems—For the construction of the C-terminal His-tagged WavL and WavM plasmidspTOPOwavL and pTOPOwavM, the pCRT7 TOPO TA Expression Kit (Invitrogen) wasused. Genes were amplified by PCR using the oligonucleotides wavL-topo-up andwavL-topo-down and chromosomal DNA of O1 as template in the case ofwavL and wavM-topo-up and wavM-topo-down and chromosomal DNA of V194as template in the case of wavM. Purified PCR products were ligatedinto pCRT7/CT-TOPO vector according to the kit manual and transformed intoTOP10F′ E. coli cells (Invitrogen). Ampr colonieswere characterized by restriction and PCR analysis. Correct construction wasverified by DNA sequencing (data not shown). Construction of His-tagged WaaLO1—For theconstruction of the N-terminal His-tagged WaaLO1 plasmidpQE30waaLO1 the gene was amplified by PCR using theoligonucleotides waaL-start-KpnI and waaL-stop-KpnI and chromosomal DNA of O1as template. The PCR product was treated with KpnI and ligated into theKpnI-digested pQE30 (Qiagen). After transformation of the ligation productsinto XL1-blue ampR colonies were obtained, isolated, and plasmidDNA was characterized by restriction analysis (data not shown). To decreasethe expression of WaaLO1 in XL1-blue Glc (0.2%) was added into theLB medium. Correct construction was verified by sequencing (data not shown).To test the expression of WaaLO1, pQE30waaLO1 wastransformed into MO10ΔwaaL. Complementation was observed bysupplementing the media with 1 mmisopropyl-β-d-thiogalactopyranoside. Expression was verifiedby immunoblot using anti-His monoclonal antibodies (Invitrogen). Construction of Amino Acid Exchanges in WaaLO1 andWaaLSARC6— Using the QuikChange site-directedmutagenesis kit (Stratagene, La Jolla, CA) defined amino acids were exchanged.According to the kit manual PCRs were carried out using pQE30waaLO1as template and the oligonucleotides waaL-R186M and waaL-R186M-antisense toswitch Arg186 to Met, waaL-H309A and waaL-H309A-antisense to switchHis309 to Ala, waaL-H311A and waaL-H311A-antisense to switchHis311 to Ala, and waaL-H309A/H311A and waaL-H309A/H311A-antisenseto switch His309 and His311 to Ala, respectively. Tolose the remaining intact plasmid template, the reaction mix was treated withDpnI, then the remaining PCR products were transformed into XL1-blue.Subsequently, ampr transformants were obtained, isolated, andplasmid DNA was prepared. The amino acids exchanges in waaL wereverified by DNA sequencing. To test for expression and complementation ofWaaLO1 -R186M, -H309A, -H311A, and -H309A/H311A, the respectiveplasmids pQE30waaLO1-R186M, -H309A, -H311A, and -H309A/H311A weretransformed into MO10ΔwaaL. For the construction of amino acidsexchange in WaaLSARC6 for Arg208 → Met andHis321 → Ala, we followed the same procedure as describedabove. There we were using plasmid pWQ322 as template and oligonucleotideswaaLSal-R208M, waaLSal-R208M-antisense, and waaLSal-H321A,waaLSal-H321A-antisense for Arg208 → Met and His321→ A exchange (Table III),respectively. Expression was verified by immunoblot using anti-His monoclonalantibodies (Invitrogen), and LPS was prepared and analyzed as describedbelow.Table IIIOligonucleotides used in this studyOligonucleotide nameOligonucleotidesequenceaRestriction sites are underlinedwavA-SacI-intern5′-AATGAGCTCAACCTGTTTTCGACTATGTAT-3′wavA-XbaI-intern5′-TATTCTAGAGATTATTCGCTGAACCACCACT-3′wavK-SacI-intern5′-TCTATCGAGCTCAATGGCAACAA-3′wavK-XbaI-intern5′-AAATCTAGAACCTATATAAATACTTTTAGCCGGA-3′wavI-SacI-intern5′-TTAGAGCTCACTACCGGTTTACCTTAACTAT-3′wavI-XbaI-intern5′-AATTCTAGATCTCGGTATTATTTTTAATGCGA-3′wavG-SacI-intern5′-TTTGAGCTCAACTTGGCATTAAACAGTT-3′wavG-XbaI-intern5′-TTTTCTAGATATTCTAAGCGATAGGAAAT-3′wavF-SacI-intern5′-AAAGAGCTCAACTTGGCATTAAACAGTT-3′wavF-XbaI-intern5′-TTTTCTAGATTAAAGTGATAAAGCCCGGT-3′wavE-SacI-intern5′-TTAGAGCTCAGAATTATCAATTTTTGACT-3′wavE-XbaI-intern5′-TTTTCTAGAAGCGCTCAGTGACATCCAAT-3′wavL-XbaI-N-term5′-TTTTCTAGAGATAACTGGCTTAGGATTATT-3′wavM-V194-SacI-intern5′-TTGAGCTCCCCATCCACTCTTTGCCA-3′wavM-V194-XbaI-intern5′-TATCTAGAAAACAAACTACATTTACCAACTT-3′waaL-V194-SacI-intern5′-TAGAGCTCCTCGCACATCAGTCATAATAAT-3′waaL-V194-XbaI-intern5′-TATTTTATCTAGATTGATTCCTATTGT-3′wavL-SacI-intern5′5′-ATGAGCTCACTCGCAGTGG-3′wavL-BamHI-intern5′5′-AAGGATCCACTGAGTCGGCCAATGATA-3′wavL-BamHI-intern3′5′-TAGGATCCAGCAAATCCCCGCTTTGA-3′wavL-BamHI-intern1-3′5′-TTAGGATCCCTTTTTGAGTAAGCTCAGTGAAT-3′wavL-XbaI-intern3′5′-AATCTAGAGTCATGTAACGCTTTAACTT-3′wavL-5′-EcoRI5′-AAGAATTCCAAAAGAGAATATGTAAGAAA-3′wavL-3′-BamHI5′-AAGGATCCGGTGTAATTAAAAGTAGTGAGA-3′wavL-N-term-up5′-AGGAGATCTTTGAAGGTCAGAGT-3′waaL-V194-5′-SacI5′-TAGAGCTCCGAGTGCCACTTTATAGAA-3′waaL-V194-3′-KpnI5′-TAGGTACCGATAACTCAGCAGCAGCAGGCAA-3′wavM-V194-5′-SacI5′-TTGAGCTCTAATCTGACCCAGTTTGATAAT-3′waaL-O1-5′-NheI5′-CTAGCAGCTAGCATTAGTTGGAACACGACCCT-3′waaL-O1-3′-KpnI5′-AAGGTACCATATCGCCAACCCAAGAAGAAGG-3′waaL-O1-up-NcoI5′-TTCCATGGTTTGCTTAAATTTTATTTTTCTTACATTGACGA-3′waaL-O1-down-NcoI5′-TTCCATGGTTTTTACTCTAATCAGTTTTTTATTA-3′waaL-V194-5′NheI5′-TATGCTAGCCGAGTGCCACTTTATAGAAAACCGA-3′waaL-V194-up-NcoI5′-TTCCATGGCATAATATTTCCATCTCAGTTTAATTGT-3′waaL-V194-down-NcoI5′-TTCCATGGTGACACTATCCGTTACTGTGCTCTCT-3′wavL-topo-up5′-ATGAATATTTTGATGGCCCTATCCCAA-3′wavL-topo-down5′-TTTTTTGTTCATTTTTCTAAATAGATAATTCCCT-3′wavM-topo-up5′-ATGAAAATCTACGTAATTAGCCTTAA-3′wavM-topo-down5′-TTTTTTATATTCATTAGCAAACATCATTAT-3′phoA-seq5′-GCTCACCAACTGATAACCAC-3′lacZ-seq5′-CCTCTTCGCTATTACGCCAG-3′waaL-start-KpnI5′-AAAGGTACCATGAATAATAAAATAACTAAAACCTCA-3′waaL-stop-KpnI5′-TATGGTACCTCAATTTTTTGCTCTTTTGGCTGCGAT-3′waaL-H311A5′-GTGCCCAGAGGTCATGCAGCCAGCCAGTATTTTGAAGCTAT-3′waaL-H311A-antisense5′-ATAGCTTCAAAATACTGGCTGGCTGCATGACCTCTGGGCAC-3′waaL-R186M5′-GCGACAATCTTAACCTTAACAATGGGAGCCATTTTAACGCTAC-3′waaL-R186M-antisense5′-GTAGCGTTAAAATGGCTCCCATTGTTAAGGTTAAGATTGTCGC-3′waaL-H309A5′-CACGGTGCCCAGAGGTGCCGCACATAGCCAGTATTTT-3′waaL-H309A-antisense5′-AAAATACTGGCTATGTGCGGCACCTCTGGGCACCGTG-3′waaL-H309A/H311A5′-CACGGTGCCCAGAGGTGCCGCAGCCAGCCAGTATTTTGAAGCTAT-3′waaL-H309A/H311A-antisense5′-ATAGCTTCAAAATACTGGCTGGCTGCGGCACCTCTGGGCACCGTG-3′waaL-Sal-H321A5′CCTTTAAAGAATCTATCGGTCCGGCAAATACCATTCTGTACATCTGGTT-3′waaLSal-H321A-antisense5′-AACCAGATGTACAGAATGGTATTTGCCGGACGATAGATTCTTTAAAGG-3′waaLSal-R208M5′-CCTGGGAACCCTATCGATGGGGGCATGGTTGGC-3′waaL-Sal-R208M-antisense5′-GCCAACCATGCCCCCATCGATAGGGTTCCCAGG-3′a Restriction sites are underlined Open table in a new tab Purification of His-tagged WavL and WavM—RecombinantHistagged WavL and WavM proteins were expressed in BL21 (DE3) pLysS asC-terminal V5 epitope/His6 fusion proteins. After transformation ofthe plasmids into BL21 (DE3) pLysS, the bacterial cells were grown to anabsorbance at 600 nm of 0.8 at 37 °C under vigorous shaking. Afterdecreasing the temperature to 20 °C the cultures were induced with 1mm isopropyl-β-d-thiogalactopyranoside andincubated overnight. For protein purification cells were harvested,resuspended in 1.5 ml of LEW buffer (50 mmNaH2PO4, 300 mm NaCl, pH 8) with proteaseinhibitor mix (Complete EDTA-free, Roche Applied Science). Cell extract wasobtained by a cell shredder (FastPrep FP120, SAVANT, Holbrook, NY) usingglass/plastic beads (Lysing Matrix B, Q BioGene) for shearing. Extracts werecentrifuged at 5,000 rpm for 10 min at 4 °C to remove intact cells andglass/plastic
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