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• W2001893708 abstract "Bordetella bronchiseptica is a pathogen of humans and animals that colonizes the respiratory tract. It produces a lipopolysaccharide O antigen that contains a homopolymer of 2,3-dideoxy-2,3-diacetamido-l-galacturonic acid (l-GalNAc3NAcA). Some of these sugars are found in the uronamide form (l-GalNAc3NAcAN), and there is no discernible pattern in the distribution of amides along the chain. A B. bronchiseptica wbmE mutant expresses an O polysaccharide unusually rich in uronamides. The WbmE protein localizes to the periplasm and catalyzes the deamidation of uronamide-rich O chains in lipopolysaccharide purified from the mutant, to attain a wild-type uronamide/uronic acid ratio. WbmE is a member of the papain-like transglutaminase superfamily, and this categorization is consistent with a deamidase role. The periplasmic location of WbmE and its acceptance of complete lipopolysaccharide as substrate indicate that it operates at a late stage in lipopolysaccharide biosynthesis, after polymerization and export of the O chain from the cytoplasm. This is the first report of such a modification of O antigen after assembly. The expression of wbmE is controlled by the Bordetella virulence gene two-component regulatory system, BvgAS, suggesting that this deamidation is a novel mechanism by which these bacteria modify their cell surface charge in response to environmental stimuli. Bordetella bronchiseptica is a pathogen of humans and animals that colonizes the respiratory tract. It produces a lipopolysaccharide O antigen that contains a homopolymer of 2,3-dideoxy-2,3-diacetamido-l-galacturonic acid (l-GalNAc3NAcA). Some of these sugars are found in the uronamide form (l-GalNAc3NAcAN), and there is no discernible pattern in the distribution of amides along the chain. A B. bronchiseptica wbmE mutant expresses an O polysaccharide unusually rich in uronamides. The WbmE protein localizes to the periplasm and catalyzes the deamidation of uronamide-rich O chains in lipopolysaccharide purified from the mutant, to attain a wild-type uronamide/uronic acid ratio. WbmE is a member of the papain-like transglutaminase superfamily, and this categorization is consistent with a deamidase role. The periplasmic location of WbmE and its acceptance of complete lipopolysaccharide as substrate indicate that it operates at a late stage in lipopolysaccharide biosynthesis, after polymerization and export of the O chain from the cytoplasm. This is the first report of such a modification of O antigen after assembly. The expression of wbmE is controlled by the Bordetella virulence gene two-component regulatory system, BvgAS, suggesting that this deamidation is a novel mechanism by which these bacteria modify their cell surface charge in response to environmental stimuli. Bordetella bronchiseptica is a Gram-negative coccobacillus, which colonizes the mammalian respiratory tract. It has a broad host range and is commonly associated with atrophic rhinitis in pigs (1Chanter N. Magyar T. Rutter J.M. Res. Vet. Sci. 1989; 47: 48-53Crossref PubMed Google Scholar) and infectious tracheobronchitis (kennel cough) in dogs (2Wagener J.S. Sobonya R. Minnich L. Taussig L.M. Am. J. Vet. Res. 1984; 45: 1862-1866PubMed Google Scholar). Most of the genes implicated in host colonization and virulence are under the transcriptional control of the two-component regulatory system BvgAS (reviewed in Ref. 3Mattoo S. Cherry J.D. Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (842) Google Scholar), being expressed maximally in the Bvg+ phase. Transcription of some other genes, for example the flagellin gene flaA, is up-regulated in Bvg– conditions (4Akerley B.J. Monack D.M. Falkow S. Miller J.F. J. Bacteriol. 1992; 174: 980-990Crossref PubMed Google Scholar) and an intermediate expression pattern (Bvgi) has also been described (5Cotter P.A. Miller J.F. Mol. Microbiol. 1997; 24: 671-685Crossref PubMed Scopus (141) Google Scholar). In vitro the Bvg– phase can be induced by culturing Bordetella with millimolar concentrations of magnesium sulfate (5Cotter P.A. Miller J.F. Mol. Microbiol. 1997; 24: 671-685Crossref PubMed Scopus (141) Google Scholar, 6van den Akker W.M. Microbiology. 1998; 144: 1527-1535Crossref PubMed Scopus (39) Google Scholar) among other stimuli. One of the Bvg-regulated bacterial structures is lipopolysaccharide (LPS). 3The abbreviations used are: LPS, lipopolysaccharide; a.m.u., atomic mass units; GalNAc, 2-acetamido-2-deoxy galactose; l-GalNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid; l-GalNAc3NAcAN, 2,3-diacetamido-2,3-dideoxy-l-galacturonamide; ManNAc3NAcAN, 2,3-diacetamido-2,3-dideoxy mannuronamide; PBS, phosphate-buffered saline; UDP-l-GalNAc3NAcA, UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid.3The abbreviations used are: LPS, lipopolysaccharide; a.m.u., atomic mass units; GalNAc, 2-acetamido-2-deoxy galactose; l-GalNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid; l-GalNAc3NAcAN, 2,3-diacetamido-2,3-dideoxy-l-galacturonamide; ManNAc3NAcAN, 2,3-diacetamido-2,3-dideoxy mannuronamide; PBS, phosphate-buffered saline; UDP-l-GalNAc3NAcA, UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. LPS is the major component of the outer leaflet of the outer membrane. LPS has three domains: first, Lipid A is the lipophilic domain that anchors LPS into the outer membrane; second, a complex, branched-chain oligosaccharide known as core is attached to lipid A and in Bordetella the lipid A-core structure is known as B-band LPS; third, a domain distal to the membrane consisting of saccharide repeats may be present, which is commonly called O antigen. In a proportion of B. bronchisepica LPS molecules the lipid A core is substituted with a trisaccharide, and this species is known as A-band LPS. The O polysaccharide consists of a homopolymer of 2,3-dideoxy-2,3-diacetamido-l-galacturonic acid (l-GalNAc3NAcA) (7Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (74) Google Scholar) capped at the nonreducing terminus with a complex 2,3,4-trideoxy-2,3,4-triamino galacturonamide (GalN3N4NAN) derivative (8Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (26) Google Scholar), and in B. bronchiseptica is attached to the A-band trisaccharide via a pentasaccharide linker (Fig. 1) (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Expression of O antigen by B. bronchiseptica is required for full virulence in animal models of infection and for resistance to complement-mediated killing (10Burns V.C. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. Infect. Immun. 2003; 71: 86-94Crossref PubMed Scopus (52) Google Scholar). A proportion of the O polysaccharide repeating units are present as the uronamide (l-GalNAc3NAcAN) (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In Bvg– B. bronchiseptica RB50, the uronamides make up ∼17% of the O polysaccharide residues and the positions of these along the chain appears to be random (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Uronamide sugars are uncommon in bacteria, but are present in the O polysaccharides of Shigella dysenteriae (11Feng L. Tao J. Guo H. Xu J. Li Y. Rezwan F. Reeves P. Wang L. Microb. Pathog. 2004; 36: 109-115Crossref PubMed Scopus (32) Google Scholar), Francisella spp (12Vinogradov E. Conlan W.J. Gunn J.S. Perry M.B. Carbohydr. Res. 2004; 339: 649-654Crossref PubMed Scopus (43) Google Scholar, 13Vinogradov E.V. Shashkov A.S. Knirel Y.A. Kochetkov N.K. Tochtamysheva N.V. Averin S.F. Goncharova O.V. Khlebnikov V.S. Carbohydr. Res. 1991; 214: 289-297Crossref PubMed Scopus (70) Google Scholar), and in Pseudomonas aeruginosa. O antigen synthesis is encoded in B. bronchiseptica by the wbm locus that contains 24 coding sequences including putative genes for the wzm and wzt components of an ATP-binding cassette (ABC) O antigen transporter (14Preston A. Allen A.G. Cadisch J. Thomas R. Stevens K. Churcher C.M. Badcock K.L. Parkhill J. Barrell B. Maskell D.J. Infect. Immun. 1999; 67: 3763-3767Crossref PubMed Google Scholar). The presence of ABC transporter genes suggests that this O antigen is probably assembled according to the ABC transporter-dependent model (reviewed in Ref. 15Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar) in which the polymer is assembled and terminated on the cytoplasmic face of the inner membrane, then exported across this membrane to the periplasmic face where the O chain is transferred to lipid A core. In 1999, Makarova et al. (16Makarova K.S. Aravind L. Koonin E.V. Protein Sci. 1999; 8: 1714-1719Crossref PubMed Scopus (142) Google Scholar) identified the B. bronchiseptica gene product WbmE as a member of the papain-like transglutaminase superfamily although in their report, wbmE was mistakenly identified as a B. pertussis sequence. Alignment of WbmE with transglutaminase conserved domains (17Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar, 18Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler G.H. Mazumder R. Nikolskaya A.N. Panchenko A.R. Rao B.S. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (648) Google Scholar) indicates that WbmE residues Cys-165, His-201, and Asp-216 probably constitute a conserved transglutaminase-type catalytic triad (19Pedersen L.C. Yee V.C. Bishop P.D. Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (137) Google Scholar) (Fig. 2). Transglutaminase activity is defined as bridge formation between peptide chains by an acyl transfer reaction between a glutamine γ-carboxamide and a lysine ϵ-amine, but transglutaminase enzymes also catalyze a range of other chemical reaction types, all of which involve either the formation, or breaking of amide bonds (reviewed in Ref. 20Lorand L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1193) Google Scholar). To date, the only functionally characterized microbial members of this family, PeiP and PeiW, are peptidases (21Luo Y. Pfister P. Leisinger T. Wasserfallen A. FEMS Microbiol. Lett. 2002; 208: 47-51Crossref PubMed Google Scholar). In this report, we describe the characterization of the wbmE gene and its protein product. WbmE catalyzes deamidation of complete O chains, and this is the first report of such a late O antigen modification. Furthermore, given that wbmE expression is regulated by the BvgAS system (22Cummings C.A. Bootsma H.J. Relman D.A. Miller J.F. J. Bacteriol. 2006; 188: 1775-1785Crossref PubMed Scopus (119) Google Scholar), this enzyme probably constitutes a novel mechanism by which the B. bronchisepica cell surface is modified in response to environmental stimuli. Bacterial Strains, Plasmids, and Culture Conditions—Bacterial strains used in this study are described in the supplemental Table S1. B. bronchiseptica was grown on Bordet-Gengou agar (Difco) supplemented with 10% defibrinated horse blood (TCS Cellworks Ltd). Escherichia coli was cultured in Luria-Bertani (LB) broth or on LB agar. All strains were incubated at 37 °C and ampicillin (100 μg ml–1), kanamycin (50 μgml–1), tetracycline (10 μg ml–1 for E. coli, 5 μg ml–1 for B. bronchiseptica) or streptomycin (200 μgml–1) were added where required. Suicide plasmids were based on the host-restricted pEX100T backbone (23Schweizer H.P. Hoang T.T. Gene. 1995; 158: 15-22Crossref PubMed Scopus (315) Google Scholar) and broad host-range shuttle vectors were based on a kanamycin resistant derivative of pBBR1MCS (24Antoine R. Locht C. Mol. Microbiol. 1992; 6: 1785-1799Crossref PubMed Scopus (202) Google Scholar). The phoA reporter fusion was derived from pRMCD28 (25Daniels C. Vindurampulle C. Morona R. Mol. Microbiol. 1998; 28: 1211-1222Crossref PubMed Scopus (157) Google Scholar). For preparation of LPS, B. bronchiseptica was grown in tryptone soya broth (Oxoid) supplemented with 50 mm MgSO4 as this maximizes O antigen expression in RB50 by modulating the phase to Bvg– (supplemental Fig. S1). DNA Methods—Standard methods were used for DNA manipulations. Oligonucleotides were supplied by Sigma-Genosys. PCR was performed with template from boiled bacteria (26Preston A. Maxim E. Toland E. Pishko E.J. Harvill E.T. Caroff M. Maskell D.J. Mol. Microbiol. 2003; 48: 725-736Crossref PubMed Scopus (71) Google Scholar) and TaqDNA polymerase (Promega) or KOD Hot Start DNA Polymerase (Novagen). Generation of wbmE Mutants—The wbmE mutant allele was obtained by in vitro transposon-mediated mutagenesis of the wbm locus-containing cosmid, BbLPS1 (14Preston A. Allen A.G. Cadisch J. Thomas R. Stevens K. Churcher C.M. Badcock K.L. Parkhill J. Barrell B. Maskell D.J. Infect. Immun. 1999; 67: 3763-3767Crossref PubMed Google Scholar) (GenBank™ accession number AJ007747) using an EZ-Tn5™ <Tet-1> insertion kit (Epicenter). The <Tet-1> transposon, plus flanking wbmE DNA, was cut out by partial digestion with AluI and ligated into SmaI-cut pEX100T. Allelic exchange constructs were transferred to B. bronchiseptica by conjugation with E. coli SM10λpir as donor (27Simon R. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5562) Google Scholar). Loss of the plasmid-encoded sacB gene in allelic exchange mutagenesis of B. bronchiseptica was selected for by growth on LB agar with reduced salt supplemented with 10% (w/v) sucrose (28Gay P. Le Coq D. Steinmetz M. Berkelman T. Kado C.I. J. Bacteriol. 1985; 164: 918-921Crossref PubMed Google Scholar). Double recombination was confirmed by Southern blotting (not shown). Multiple mutant clones were obtained in independent mating experiments, and were confirmed to have the same phenotype as the representative wbmE strain, RBE3c (not shown). Complementation of wbmE Mutation—The B. bronchiseptica flaA promoter was amplified using primers 5′-GCTCTAGATAGGCGCATGCCATGGCC-3′ (XbaI site underlined) and 5′-AAGGATCCCATATGGAGGCTCCCAAGAGAGAA-3′ (BamHI and NdeI sites underlined), and cloned into the XbaI and BamHI sites in pBBR1MCS-kan to generate the vector pCompEmpty. wbmE was amplified using primers 5′-AAAAAAACATATGATTCGCAAGAGCTAC-3′ (NdeI site underlined) and 5′-AAAAAGCTTAGATCTCCACATAGAGCAGATGTC-3′ (HindIII site underlined) and topoisomerase-cloned into pCR2.1-TOPO, and the insert was verified by sequencing. wbmE was then excised and cloned into pCompEmpty using NdeI and HindIII restriction sites to generate the wbmE complementation vector pCompE. For complementation of the wbmE mutation by expressing WbmE with a C-terminal His6 tag, the pCR2.1-TOPO vector containing wbmE was used as PCR template with the primers 5′-CCCGGTTTGAAGAAGCCTTTCTC-3′ and 5′-AAAGCTTCAGTGATGATGATGATGATGGTTCGGGGCGCTGGCGCG-3′ (HindIII site underlined, reverse complement of His6 codons italicized). wbmE-his6 was then cloned into pCompEmpty using NdeI and HindIII, generating pCompETag. Shuttle vectors were moved into B. bronchiseptica by conjugation with E. coli CC118 as donor (29Manoil C. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8129-8133Crossref PubMed Scopus (655) Google Scholar), with trans-acting transfer functions provided by E. coli S17-1 pNJ5000 as helper (27Simon R. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5562) Google Scholar, 30Grinter N.J. Brewster G. Barth P.T. Plasmid. 1989; 22: 203-214Crossref PubMed Scopus (18) Google Scholar). SDS-PAGE Analysis of LPS—LPS for SDS-PAGE analysis was obtained from B. bronchiseptica using a modification of the method of Hitchcock and Brown (31Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar) as has been described (32Preston A. Maskell D. Johnson A. Moxon E.R. J. Bacteriol. 1996; 178: 396-402Crossref PubMed Google Scholar). SDS-PAGE of LPS was performed using Novex precast 16% Tricine gels (Invitrogen). LPS was oxidized in-gel with periodic acid (33Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2279) Google Scholar) and visualized with the Silver Stain Plus kit (Bio-Rad). Purification of LPS—B. bronchiseptica RB50 and RBE3c LPS were extracted from 4-liter cultures using a modification of the hot aqueous phenol extraction method of Johnson and Perry (34Johnson K.G. Perry M.B. McDonald I.J. Russel R.R. Can. J. Microbiol. 1975; 21: 1969-1980Crossref PubMed Scopus (20) Google Scholar) as has been described (26Preston A. Maxim E. Toland E. Pishko E.J. Harvill E.T. Caroff M. Maskell D.J. Mol. Microbiol. 2003; 48: 725-736Crossref PubMed Scopus (71) Google Scholar). Analysis of LPS Structures—O polysaccharides were cleaved from LPS molecules by 24-h solvolysis with anhydrous HF and subsequently purified as described (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Electrospray ionization MS spectra were obtained on a Micromass Quattro spectrometer, with samples dissolved in 50% MeCN, 0.2% HCOOH, and delivered by direct injection at a flow rate of 15 μl min–1. Capillary electrophoresis-mass spectrometry (CE-MS) experiments were performed as described previously (35Li J. Richards J.C. Mass. Spectrom. Rev. 2007; 26: 35-50Crossref PubMed Scopus (48) Google Scholar). Generation of the phoA Fusion and Alkaline Phosphatase Assay—The first 200 bases of wbmE were PCR-amplified using the primers 5′-AAAAAAAGATCTCCGCGGAAGGAGGATATACATATGATTCGCAAGAGCTACATCATCG-3′ (SacII site underlined) and 5′-AAAAAAAAGCTTTTAAAGGATCCAGAATTCCAGAAGGCGTAGCAAGTCCGGC-3′ (HindIII site underlined). The PCR product was digested with HindIII and SacII, and cloned into similarly digested pRMCD28 (25Daniels C. Vindurampulle C. Morona R. Mol. Microbiol. 1998; 28: 1211-1222Crossref PubMed Scopus (157) Google Scholar), placing the wbmE 5′-end in-frame with the phoA fragment on the vector. The reporter plasmid was transformed into the phoA E. coli strain LMG194. For alkaline phosphatase assays, 5 μl of stationary phase liquid culture was spotted onto low-phosphate solid medium (120 mm Tris-HCl, pH 7.4, 40 mm NaCl, 20 mm KCl, 40 mm NH4Cl, 20 mm Na2SO4, 1 mm MgCl2, 0.2 mm CaCl2, 0.004 mm ZnCl2, 0.002 mm FeCl3, 0.1 mm KH2PO4, 0.4% (w/v) glycerol, 1.5% (w/v) agar) supplemented with 40 μgml–1 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), 1 mm isopropyl β-d-1-thiogalactopyranoside (IPTG), 5 μgml–1 thiamine, 0.5% (w/v) casamino acids, and ampicillin) and incubated for 16 h at 37 °C. Expression of WbmE-His6 in B. bronchiseptica—The plasmid pCompETag has the wbmE-his6 gene fusion under the control of the promoter for the B. bronchiseptica flagellin gene flaA. As flaA is expressed in the Bvg– phase (36Akerley B.J. Miller J.F. J. Bacteriol. 1993; 175: 3468-3479Crossref PubMed Google Scholar), WbmE-His6 expression from this vector was induced by supplementing the medium with 50 mm MgSO4. WbmE Assay using Whole Cell Lysates—For analysis by SDS-PAGE: B. bronchisepica was harvested from 75-ml liquid cultures at an absorbance of 0.2 (at 595 nm). Cells were washed with 37.5 ml of phosphate-buffered saline at pH 6.5 (PBS), and suspended in 5 ml of PBS. One-tenth and one-hundredth dilutions were made of these suspensions. 1:1 mixtures were made of these neat, one-tenth, and one-hundredth cell suspensions with a 0.4 mg ml–1 solution of purified RBE3c LPS dissolved in PBS. WbmE was then released from the cells by sonicating the mixtures on ice for 15 s, using a VibraCell™, sonicator (Sonics and Materials Inc.), fitted with a microtip, at 40% power. They were then incubated at 37 °C for 3, 30, or 300 min with shaking. Samples were centrifuged at 15,000 × g for 5 min at 4 °C, and 100 μl of the supernatant was boiled with 50 μl of buffer 1 (0.1875 m Tris-HCl, pH 6.8, 6% (w/v) SDS, 30% (w/v) glycerol), and then incubated overnight at 55 °C after addition of 225 μl of buffer 2 (10 mg ml–1 proteinase K, 0.0625 m Tris-HCl, pH 6.8, 0.1% (w/v) SDS, 10% (w/v) glycerol, 0.1% (w/v) bromphenol blue). Samples were boiled prior to loading 10 μl per lane on Tricine SDS-PAGE gels. In this assay, whole cell lysates are used as a source of enzyme. So that no O antigen derived from the cells themselves could interfere with analysis of the substrate after incubation, we used a B. bronchisepica strain, RBB1a, as the wbmE+ cells. RBB1a is a B. bronchiseptica RB50-derived mutant in the putative glycosyl transferase wbmB, which does not produce O antigen. 4J. D. King, A. Preston, and D. J. Maskell, unpublished results. To maximize the amount of WbmE in the lysates, the vector pCompETag was also maintained in these RBB1a cells so that the enzyme is probably expressed from the plasmid as well as from the chromosomal copy of the wbmE gene. For the negative control, whole cell lysates were obtained from the wbmE mutant strain, RBE3c, containing the empty vector pCompEmpty. For CE-MS characterization of the transformed O antigen, incubations were performed in the same proportions, but at larger scale and for 16 h, with 15 mg of RBE3c LPS in each sample and using neat wbmE+ or wbmE– cell suspensions. Purification of WbmE-His6—WbmE-His6 was expressed in B. bronchisepica from the plasmid pCompETag as before. Cells were pelleted (10 min at 10,000 × g) from 1 liter of culture and frozen (–20 °C). The pellet was thawed and suspended in 20 ml of 50 mm Tris, pH 8.5, 300 mm NaCl. Cells were broken with ultrasound (40% power, macro-tip, 3 min, in 5-s bursts, on ice), and cell debris was pelleted (20 min at 20,000 × g). Membranes were removed by ultracentrifugation (1 h at 100,000 × g), and the supernatant was incubated with 3 ml of nickel-nitrilotriacetic acid (Ni-NTA) slurry (Qiagen) for 1 h at 4°C with gentle agitation. The nickel affinity resin was loaded into a column, washed with 25 ml of 50 mm Tris, pH 8.5, 300 mm NaCl, 50 mm imidazole, and then WbmE-His6 was eluted with small fractions of 50 mm Tris, pH 8.5, 300 mm NaCl, 200 mm imidazole. 1.6 ml of eluent containing most of the eluted protein was diluted one-sixth with water and loaded onto a 5-ml Econo-Pac High-Q anion exchange column (Bio-Rad). The column was washed with 50 ml of 20 mm Tris, pH 8.5, 50 mm NaCl. The column was eluted with a linear gradient of 50–1000 mm NaCl in 20 mm Tris, pH 8.5 over 50 ml. WbmE-His6 eluted into five 1-ml fractions at ∼200 mm NaCl. The activity of each fraction was tested by incubating overnight (37 °C) with an equal volume of 0.4 mg ml–1 RBE3c LPS dissolved in 50 mm Tris, pH 8.5, 300 mm NaCl. After incubation, the LPS was processed for analysis by SDS-PAGE as described above. WbmE-His6-containing fractions were then pooled, glycerol was added to 25% (v/v), and the protein was stored at –20 °C. Analysis of the wbmE LPS Phenotype by SDS-PAGE—To characterize the role of wbmE in O antigen expression it was mutated by insertion of a tetracycline resistance gene cassette into the coding sequence. Disruption of wbmE does not alter the A- or B-band LPS but does change the appearance of O band LPS on SDS-PAGE (Fig. 3). The mutant O band has reduced electrophoretic mobility and has a more clearly resolved banding pattern. Individual wild-type O antigen-containing species are not so clearly resolved, the O band appearing as a smear on the gel. Complementation of the wbmE mutation by expression of the wild-type allele from a plasmid restores the wild-type electrophoretic mobility of O band (Fig. 3). The mutation can also be complemented by a vector in which codons for a His6 tag are fused to the 3′-end of the wbmE gene (Fig. 3). wbmE Mutant LPS Differs from Wild-type by Having a Greater Number of Uronamides in the O Antigen—Altered electrophoretic mobility in a polymeric molecule such as O antigen often indicates that the chain length has altered (37Clarke B.R. Cuthbertson L. Whitfield C. J. Biol. Chem. 2004; 279: 35709-35718Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), but can also reflect a change in the electrostatic charge on O antigen sugars (38King J.D. Mulrooney E.F. Vinogradov E. Kneidinger B. Mead K. Lam J.S. J. Bacteriol. 2008; 190: 1671-1679Crossref PubMed Scopus (20) Google Scholar). To determine the cause of the SDS-PAGE band shift in this case, we purified LPS from the wild-type and wbmE mutant and analyzed their O chains by mass spectrometry. Prior to analysis, O antigen was cleaved from the rest of the LPS molecule by solvolysis with anhydrous hydrogen fluoride, which cleaves the polysaccharide chain at the GalNAc position in the O antigen linker region (Fig. 1) (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Electrospray mass spectra of wild-type and wbmE O polysaccharides both show a series of peaks separated by ∼258 atomic mass units (a.m.u.) (Fig. 4). Because 258 a.m.u. is approximately the mass of an O antigen GalNAc3NAcA(N) repeating unit, these series represent the variation in O chain lengths expressed by the bacteria. There was no difference in the gross distribution of chain lengths between the two samples demonstrating that the reduction in electrophoretic mobility of the mutant O antigen-containing LPS is not due to increased O chain length. Furthermore, each of the peaks in the spectrum derived from the wbmE mutant sample is shifted downwards by 4–6 a.m.u compared with corresponding peaks in the spectrum of wild-type O chain. MS and NMR analysis of wild-type O antigen has established that the O repeating units are present as both uronic acids (GalNAc3NAcA) and uronamides (GalNAc3NAcAN) (9Preston A. Petersen B.O. Duus J.O. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We hypothesized, therefore, that the difference in mass between the wbmE mutant and wild-type O polysaccharide species was due to the wbmE mutant producing a greater proportion of uronamide residues (between four and six additional uronamides per LPS molecule). An implication of this hypothesis is that the mutant will have 4–6 fewer negative charges per O antigen molecule, and this is consistent with slower migration toward the anode in SDS-PAGE. The molecular weights measured for particular peaks in the wbmE O antigen mass spectrum suggested that while this mutant expresses a more uronamide-rich O polysaccharide than its parental strain, the wbmE O antigen still contains a mixture of uronic acid and uronamide residues. For example the peak at 3383 corresponds with the predicted molecular weight of an HF-cleaved polysaccharide containing five GalNAc3NAcAN residues, four GalNAc3NAcA residues, the capping sugar, and the ManNAc3NAcAN-GalNAc3NAcAN-GalNAc portion of the linker (calculated MW = 3383.1). To confirm our interpretation of the electrospray MS data, HF-cleaved O polysaccharide was subjected to fragmentation in a capillary electrophoresis mass spectrometry (CE-MS) experiment. The pseudo-tandem mass spectra of wild-type and wbmE O polysaccharides show peaks corresponding to mono-, di-, tri-, and tetrasaccharide fragments derived from the polymers (Fig. 5, A and B). Interpretation of these spectra is complicated by the fact that true M+1 peaks due to additional uronic acids overlap with isotopic peaks, but comparison of the two spectra indicates that wbmE O antigen fractures to give a greater proportion of all-uronamide fragments, but the distribution of masses still indicates the presence of uronic acid residues in the mutant O chain. WbmE Is Localized in the Periplasm—Analysis of the structure of the LPS synthesized by the wbmE mutant suggested that WbmE plays a role in converting uronamide residues to uronic acids. Conceivably this could occur either by WbmE acting on completed O chain, or WbmE could operate at an earlier stage, and catalyze the deamidation of a sugar-nucleotide O antigen precursor. The stage at which LPS biosynthetic enzymes operate is dictated by their cellular localization: sugar-nucleotides are soluble, cytoplasmic metabolites, and according to the ABC transporter-dependent model of O antigen biosynthesis, the O polysaccharide will be completed before it is transported across the inner membrane. Analysis of the WbmE sequence using the LipoP 1.0 signal peptide prediction server (39Juncker A.S. LipoP 1.0 Signal Peptide Prediction Server. 2003; (Technical University of Denmark)Google Scholar, 40Juncker A.S. Willenbrock H. Von Heijne G. Brunak S. Nielsen H. Krogh A. Protein Sci. 2003; 12: 1652-1662Crossref PubMed Scopus (875) Google Scholar) predicts a signal peptidase I cleavage signal (log-odds score >10) including a predicted transmembrane helix close to the N terminus (Ile-7 to Gln-26) with the peptide bond targeted for cleavage probably one of those between amino acids Gly-21 and Ala-32. WbmE is therefore highly likely to be secreted from the cytoplasm. To verify the function of the predicted signal peptide, and localize WbmE, C-terminally His6-tagged WbmE (WbmE-His6) expression was induced in B. bronchiseptica from the vector previously used to test the ability of" @default.
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• W2001893708 title "Post-assembly Modification of Bordetella bronchiseptica O Polysaccharide by a Novel Periplasmic Enzyme Encoded by wbmE" @default.
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