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• W2011574741 abstract "N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures ofPseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis ofN-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype. N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures ofPseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis ofN-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype. lipopolysaccharide capillary electrophoresis capsular polysaccharide heteronuclear multiple bond correlation heteronuclear single quantum coherence monoclonal antibody mass spectroscopy nuclear Overhauser enhancement NOE spectroscopy Short Chain Dehydrogenase/Reductase total correlation spectroscopy uridine diphosphate UDP-2-acetamido-2,6-dideoxy-l-galactose or UDP-N-acetyl-l-fucosamine UDP-2-acetamido-2-deoxy-d-glucose or UDP-N-acetyl-d-glucosamine UDP-2-acetamido-2-deoxy-d-mannose or UDP-N-acetyl-d-mannosamine 2-acetamido-2,6-dideoxy-l- talose Surface carbohydrate structures are important virulence factors of Gram-positive and Gram-negative bacteria. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa causes fatal lung infections in patients suffering from the inherited disorder cystic fibrosis; it also affects burn victims and immunocompromised individuals undergoing anticancer chemotherapy and those infected with human immunodeficiency virus. This bacterium produces a variety of polysaccharide structures, one of which is lipopolysaccharide (LPS).1 LPS is composed of three distinct units: (i) lipid A, which noncovalently binds the molecule into the outer membrane, (ii) the core region that links, and (iii) the O antigen, composed of linear repeats of di- to hexasaccharide units, to the lipid A anchor (1Raetz C.R. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar). In the case ofP. aeruginosa, two different O antigens are made, namely A-band and B-band (2Rocchetta H.L. Burrows L.L. Lam J.S. Microbiol. Mol. Biol. Rev. 1999; 63: 523-553Crossref PubMed Google Scholar). A-band is a homopolymer ofd-rhamnose present in most serotypes, whereas B-band is a serospecific heteropolymer consisting of di- to pentasaccharide repeating units. Differences in B-band repeating units are responsible for the classification into 20 International Antigenic Typing System serotypes (3Liu P.V. Matsumoto H. Kusama H. Bergan T. Int. J. Sys. Bacteriol. 1983; 33: 256-264Crossref Scopus (132) Google Scholar, 4Liu P.V. Wang S. J. Clin. Microbiol. 1990; 28: 922-925Crossref PubMed Google Scholar). Mutants devoid of O antigen are 1000-fold less virulent than wild-type bacteria (5Cryz S.J., Jr. Pitt T.L. Furer E. Germanier R. Infect. Immun. 1984; 44: 508-513Crossref PubMed Google Scholar). Staphylococcus aureus is an important bacterial pathogen responsible for a broad spectrum of human and animal diseases including cutaneous as well as wound infections and more life-threatening infections such as endocarditis and bacteremia. Moreover, S. aureus produces numerous exotoxins, some of which cause diseases such as toxic shock syndrome and food poisoning (6Lowy F.D. N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4673) Google Scholar). The majority of clinical S. aureus isolates produce either a type 5 or type 8 capsule (CP), which renders the organisms resistant to phagocytic uptake (7Thakker M. Park J.S. Carey V. Lee J.C. Infect. Immun. 1998; 66: 5183-5189Crossref PubMed Google Scholar). Moreover, CP production has been shown to enhance virulence in animal models of infection (8Portoles M. Kiser K.B. Bhasin N. Chan K.H. Lee J.C. Infect. Immun. 2001; 69: 917-923Crossref PubMed Scopus (49) Google Scholar). S. aureus is highly efficient at acquiring resistance to antibiotics; the first documented case of a vancomycin-resistant S. aureus infection in a United States patient was recently reported by the Centers for Disease Control (60Sievert D.M. Boulton M.L. Stoltman G. Johnson D. Stobierski M.G. Downes F.P. Somsel P.A. Rudrik J.T. Hafeez W. Lundstrom T. Flanagan E. Johnson R. Mitchell J. Chang S. Morbidity and Mortality Weekly Report. 51. Centers for Disease Control, Atlanta2002: 565-567Google Scholar). l-FucNAc has thus far been described exclusively as a constituent of bacterial polysaccharide structures. It is part of the O antigen of P. aeruginosa serotypes O4, O11, and O12, of the CP of S. aureus serotypes 5 and 8, and ofStreptococcus pneumoniae CP type 4 (9Knirel Y.A. Kochetkov N.K. Biochemistry (Moscow). 1994; 59: 1325-1383Google Scholar, 10Moreau M. Richards J.C. Fournier J.M. Byrd R.A. Karakawa W.W. Vann W.F. Carbohydr. Res. 1990; 201: 285-297Crossref PubMed Scopus (75) Google Scholar, 11Fournier J.M. Vann W.F. Karakawa W.W. Infect. Immun. 1984; 45: 87-93Crossref PubMed Google Scholar, 12Jones C. Currie F. Forster M.J. Carbohydr. Res. 1991; 221: 95-121Crossref PubMed Scopus (37) Google Scholar). LPSs ofEscherichia coli O26 and CP of Bacteroides fragilis also contain l-FucNAc (13Manca M.C. Weintraub A. Widmalm G. Carbohydr. Res. 1996; 281: 155-160Crossref PubMed Scopus (24) Google Scholar, 14Kasper D.L. Weintraub A. Lindberg A.A. Lonngren J. J. Bacteriol. 1983; 153: 991-997Crossref PubMed Google Scholar). It should be noted that not all l-FucNAc-containing bacteria are listed above; for the scope of this study, only those bacteria for which sequence data of the corresponding polysaccharide biosynthesis gene clusters are available have been taken into consideration (E. coli O26 polysaccharide cluster data are referred to in Ref. 15Jiang S.M. Wang L. Reeves P.R. Infect. Immun. 2001; 69: 1244-1255Crossref PubMed Scopus (93) Google Scholar). With respect to P. aeruginosa, thel-FucNAc-containing strains, particularly International Antigenic Typing System O4 and O11, belong to the most clinically prevalent strains besides serotypes O3 and O6 (16Pitt T.L. Eur. J. Clin. Microbiol. Infect. Dis. 1988; 7: 238-247Crossref PubMed Scopus (98) Google Scholar). The O11 strain PA103 is a high level exotoxin A producer (17Pavlovskis O.R. Pollack M. Callahan III, L.T. Iglewski B.H. Infect. Immun. 1977; 18: 596-602Crossref PubMed Google Scholar). S. aureus CP serotypes 5 and 8 make up ∼80% of clinical isolates (18Sompolinsky D. Samra Z. Karakawa W.W. Vann W.F. Schneerson R. Malik Z. J. Clin. Microbiol. 1985; 22: 828-834Crossref PubMed Google Scholar), andl-FucNAc is a component of both CP structures. Moreover, both P. aeruginosa and S. aureus are associated with nosocomial infections, as well as being known for resistance against antibiotics. S. pneumoniae type 4 capsule is a component of a heptavalent streptococcal vaccine, which is available commercially (19Alpern E.R. Alessandrini E.A. McGowan K.L. Bell L.M. Shaw K.N. Pediatrics. 2001; 108: E23Crossref PubMed Scopus (47) Google Scholar). These data implicate the importance of thel-FucNAc residue in pathogenicity. Targeting its biosynthesis could lead to the development of therapeutic agents that affect important virulence factors of Gram-positive and Gram-negative bacteria. To date, the biosynthesis pathway leading to the nucleotide-activated precursor of l-FucNAc is not clearly defined. Two putative pathways have been proposed, both lacking any experimental evidence. Lee and Lee (20Lee J.C. Lee C.Y. Goldberg J.B. Genetics of Bacterial Polysaccharides. CRC Press, Boca Raton, FL1999: 185-205Crossref Google Scholar) suggested a three-step route starting from UDP-d-ManNAc and involving (i) 3-epimerization, (ii) 5-epimerization, and (iii) 6-dehydration leading to UDP-l-FucNAc. Jiang et al. (15Jiang S.M. Wang L. Reeves P.R. Infect. Immun. 2001; 69: 1244-1255Crossref PubMed Scopus (93) Google Scholar) proposed a reaction scheme analogous to the biosynthesis of GDP-l-fucose from GDP-d-mannose. This group suggested that UDP-d-GlcNAc is first converted to UDP-d-ManNAc to get the same precursor as in the above pathway. The next reaction step is 4,6-dehydration to get UDP-2-acetamido-2,6-dideoxy-d-lyxo-4-hexulose, and finally a bifunctional enzyme would catalyze 3,5-epimerization and 4-reduction to yield UDP-l-FucNAc. The S. aureusgenes cap5/8E, cap5/8F, andcap5/8G or the homologous S. pneumoniae genes cps4M(J), cps4N(K), andcps4L have been proposed to be involved in this biosynthesis (15Jiang S.M. Wang L. Reeves P.R. Infect. Immun. 2001; 69: 1244-1255Crossref PubMed Scopus (93) Google Scholar, 20Lee J.C. Lee C.Y. Goldberg J.B. Genetics of Bacterial Polysaccharides. CRC Press, Boca Raton, FL1999: 185-205Crossref Google Scholar). We present for the first time a proposed biosynthetic pathway for nucleotide-activated l-FucNAc, namely UDP-l-FucNAc from its precursor UDP-d-GlcNAc. We provide evidence that three P. aeruginosa enzymes, WbjB, WbjC, and WbjD (and their S. aureus homologs Cap5E, Cap5F, and Cap5G) catalyze a five-step reaction cascade. UDP-N-acetyl-d-glucosamine, UDP-N-acetyl-d-galactosamine, UDP-d-glucose, NAD+, NADH, NADP+, NADPH, and the antibiotics used in this study were obtained from Sigma-Aldrich. HiTrap Chelating columns were purchased from AmershamBiosciences, Econo-Pac High Q columns were from Bio-Rad, and pET28a, pET24a+, and pET24c+ were from Novagen (Madison, WI). The bacterial strains and plasmids used in this study are listed in Table I. P. aeruginosa strains were grown in Luria broth (Invitrogen) or onPseudomonas Isolation Agar (Difco Laboratories, Detroit, MI). S. aureus was grown in tryptic soy broth (Difco Laboratories). E. coli strains Top10 (Invitrogen) and JM109 (21Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar) were used for plasmid propagation. E. coli strain SM10 (22Simon R. Priefer U. Puhler A. BioTechnology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar) was used as donor in bacterial conjugation. For protein overexpression E. coli strain BL21(DE3) was used (Novagen). Overexpression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside (Invitrogen) at a final concentration of 1 mm. The media were supplemented with ampicillin (100–250 μg/ml), kanamycin (25 μg/ml or 50 μg/ml), carbenicillin (250 μg/ml for E. coli and 300 μg/ml for P. aeruginosa), gentamicin (15 μg/ml forE. coli and 300 μg/ml for P. aeruginosa), tetracycline (15 μg/ml for E. coli and 100 μg/ml forP. aeruginosa), chloramphenicol (10 μg/ml), or erythromycin (10 μg/ml) when necessary.Table IBacterial strains and plasmids used in this studyGenotype/descriptionSourceStrains E. coli Top10F− mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1araD139 Δ(araA-leu)7697galU gal K rpsL (StrR)endA1 nupGInvitrogen JM109endA1 recA1 gyrA96thi hsdR17 (r k−,m k+)relA1 supE44 λ− Δ(lac-proAB) [F′ traD36 proABlaqIqZΔM15Ref. 21Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar SM10thi-1 thr leu tonA lacY supE recA RP4–2-Tc::Mu, KmrRef. 22Simon R. Priefer U. Puhler A. BioTechnology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar BL21(DE3)E. coli B F− dcm ompT hsdS(r B− m B−) gal λ(DE3)Novagen P. aeruginosa IATS O11Wild-type strain serotype O11 (ATCC 33358)ATCC PA103Wild-type strain serotype O11Ref. 52Liu P.V. J. Infect. Dis. 1973; 128: 506-513Crossref PubMed Scopus (115) Google Scholar BK103BPA103 withwbjB::aacC1 mutationThis study S. aureus VI-114Capsular serotype 5 strainA. W. Chow (University of British Columbia) NewmanCP5-positiveNCTC 8178 G01Newman withermB-inactivated cap5G geneThis study F4Newman with ermB-inactivated cap5FgeneThis study RN4220CP-negative, restriction-negativeRef. 53Peng H.L. Novick R.P. Kreiswirth B. Kornblum J. Schlievert P. J. Bacteriol. 1988; 170: 4365-4372Crossref PubMed Scopus (420) Google Scholar 8325–4NCTC 8325 cured of prophages; CP-negativeRef. 54Novick R. Virology. 1967; 33: 155-166Crossref PubMed Scopus (531) Google ScholarPlasmids pET23derExpression vector for histidine fusionRef. 33Newton D.T. Mangroo D. Biochem. J. 1999; 339: 63-69Crossref PubMed Scopus (33) Google Scholar pET28aExpression vector for histidine fusionNovagen pET24a+Expression vector for histidine fusionNovagen pET24c+Expression vector for histidine fusionNovagen pEX100TConjugation vector withbla and sacB genesRef. 36Schweizer H.P. Hoang T.T. Gene (Amst.). 1995; 158: 15-22Crossref PubMed Scopus (319) Google Scholar pUCGmpUC derivative containing aacC1 geneRef. 55Schweizer H.D. BioTechniques. 1993; 15: 831-834PubMed Google Scholar pUCP27E. coli and P. aeruginosa shuttle plasmid for gene complementationRef. 56West S.E. Schweizer H.P. Dall C. Sample A.K. Runyen-Janecky L.J. Gene (Amst.). 1994; 148: 81-86Crossref PubMed Scopus (498) Google Scholar pFuc11wbjB in pET28a (NdeI/EcoRI)This study pFuc12wbjC in pET28a (NdeI/HindIII)This study pFuc13wbjD in pET23der (NcoI/BamHI)This study pFuc21cap5E in pET23der (NcoI/BamHI)This study pKOB1wbjB in pEX100T (SmaI)This study pKOB2Insertion of aacC1 in wbjBof pKOB1 (KpnI)This study pPAB27wbjB in pUCP26 (XbaI/EcoRI)This study pSAE27cap5E in pUCP26 (XbaI/KpnI)This study pKBK50dcap5E in pET24a+ (XhoI/NheI)This study pET5F1.1cap5F in pET24a+ (XhoI/NheI)This study pKBK6acap5G in pET24a+ (XbaI/EcoRI)This study pJCL69Subclone of cap5 (contains intactcapF through capH) in pGEM-7Zf+Ref. 57Bhasin N. Albus A. Michon F. Livolsi P.J. Park J.S. Lee J.C. Mol. Microbiol. 1998; 27: 9-21Crossref PubMed Scopus (49) Google Scholar pJCL69–2294-bp BclI fragment deleted fromcap5G in pJCL69This study pERMBermBcassette (from Tn551) in pGEM7Zf+Ref. 39Kiser K.B. Bhasin N. Deng L. Lee J.C. J. Bacteriol. 1999; 181: 4818-4824Crossref PubMed Google Scholar pJCL69–2ermB1.3-kb ermB fragment (BamHI/BclI) from pERMB in pJCL69–2This study pCL10E. coli/S. aureustemperature-sensitive shuttle vectorRef. 58Sau S. Sun J. Lee C.Y. J. Bacteriol. 1997; 179: 1614-1621Crossref PubMed Google Scholar pKOR14.4-kbXbaI/BamHI fragment from pJCL69–2ermB in pCL10This study pAP1.2EXpCL10 containing ermBcassetteThis study pKOR31.6-kb amplicon (cap5Gand 450 bp of cap5F) in pAP1.2EX (XbaI)This study pKOR41.9-kb amplicon (385 of bp cap5D, cap5E and 530 bp cap5F) in pKOR3 (SacI/EcoRV)This study pLI50E. coli/S. aureus shuttle vectorRef. 59Lee C.Y. Buranen S.L. Ye Z.H. Gene (Amst.). 1991; 103: 101-105Crossref PubMed Scopus (208) Google Scholar pJCL432.2-kb cap5 subclone in pGEM7Zf+ (contains cap5G)Ref. 57Bhasin N. Albus A. Michon F. Livolsi P.J. Park J.S. Lee J.C. Mol. Microbiol. 1998; 27: 9-21Crossref PubMed Scopus (49) Google Scholar pKOR22.2-kbBamHI/XbaI fragment of pJCL43 carryingcap5G in pLI50This study pCAP16Subclone ofcap5 locus in pCU1; contains cap5A throughcap5ERef. 44Wann E.R. Dassy B. Fournier J.M. Foster T.J. FEMS Microbiol. Lett. 1999; 170: 97-103Crossref PubMed Google Scholar pCAP17Subclone of cap5locus in pCU1; contains cap5A through cap5FT. Foster (Ireland) Open table in a new tab SDS-PAGE was done according to the method of Laemmli (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) with slight modifications. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich). The protein concentrations were determined as described by Bradford (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). LPS was prepared according to the method of Hitchcock and Brown (25Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). The samples were run on a 12% SDS-PAGE gel, and the LPS was either transferred to a nitrocellulose membrane or visualized by a rapid silver staining procedure (26Fomsgaard A. Freudenberg M.A. Galanos C. J. Clin. Microbiol. 1990; 28: 2627-2631Crossref PubMed Google Scholar). Antibodies MF55-1 (mAb to B-band O antigen of serotype O11) (27Lam J.S. MacDonald L.A. Lam M.Y. Duchesne L.G. Southam G.G. Infect. Immun. 1987; 55: 1051-1057Crossref PubMed Google Scholar), grouping antiserum O11 (polyclonal antibody to B-band O antigen of serotype O11; Chengdu Institute of Biological Products, Chengdu, China), and N1F10 (mAb to A-band O antigen) (28Lam M.Y. McGroarty E.J. Kropinski A.M. MacDonald L.A. Pedersen S.S. Hoiby N. Lam J.S. J. Clin. Microbiol. 1989; 27: 962-967Crossref PubMed Google Scholar) were used for Western immunoblotting analysis. The blots were developed with anti-mouse immunoglobulin G alkaline phosphatase (Jackson Immunoresearch, West Grove, PA) for mAbs, and anti-rabbit immunoglobulin G alkaline phosphatase (Bio-Rad) for the polyclonal antiserum. 5-Bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma-Aldrich) were used as the alkaline phosphatase substrate. Colony immunoblot analysis was performed as previously described (29Lee J.C. Liu M.J. Parsonnet J. Arbeit R.D. J. Clin. Microbiol. 1990; 28: 2612-2615Crossref PubMed Google Scholar) with the use of CP5-specific polyclonal rabbit antiserum. The blots were developed using protein A-horseradish peroxidase conjugate (Zymed Laboratories Inc., South San Francisco, CA) and the Bio-Rad horseradish peroxidase conjugate substrate kit. BLAST (30Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) and Multalin (31Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4347) Google Scholar) were used for analysis of nucleotide and protein sequences. All of the standard DNA recombinant procedures were performed according to the methods described by Sambrook et al. (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or as recommended by the corresponding manufacturer. PCR was carried out with a GeneAmp PCR System 2400 (PerkinElmer Life Sciences). DNA sequencing was performed by Mobix Lab (Hamilton, Canada) or by the Beth Israel Deaconess Molecular Medicine Unit (Boston, MA). N-terminal protein sequencing was performed by the Dana Farber Molecular Biology Core Facility (Boston, MA). The oligonucleotide primer sequences are given in Table II. Chromosomal DNA ofP. aeruginosa International Antigenic Typing System strain O11 or S. aureus capsular type 5 was used for amplification of the genes. Pwo polymerase (Roche Molecular Biochemicals) was used for PCR amplification. Primer pairs wbjB1F/wbjB2R, wbjC1F/wbjC2R, wbjD1F/wbjD2R, and capE1F/capE2R were used for amplification of thewbjB, wbjC, wbjD, and cap5E(clinical isolate provided by A. Chow, Vancouver, Canada) genes, respectively. The PCR products of genes wbjD andcap5E were digested with NcoI andBamHI and ligated into the modified pET23 derivative (33Newton D.T. Mangroo D. Biochem. J. 1999; 339: 63-69Crossref PubMed Scopus (33) Google Scholar), cut with the same enzymes. PCR product wbjC was cut withNdeI and HindIII and ligated into appropriately digested pET28a. Finally, PCR product wbjB was digested withNdeI and EcoRI and ligated intoNdeI/EcoRI-cut pET28a. The corresponding plasmids pFuc11 (wbjB), pFuc12 (wbjC), pFuc13 (wbjD), and pFuc21 (cap5E) were confirmed by DNA sequencing and used for amplification of PCR products for complementation studies and/or overexpression of N-terminal histidine-tagged fusion proteins.Table IIOligonucleotide primers used in this studyPrimer2-aForward and reverse primers are represented by plus and minus signs, respectively.Primer sequence2-bThe underlined sequences represent the restriction sites used to clone the PCR products.wbjB1F (+)5′-GGACTCGAGCATATGGATAAGAACTCTGTTC-3′wbjB2R (−)5′-GGAATTCAGTTACAAGAACTTTCATCG-3′wbjC1F (+)5′-GTAGATCTCATATGAAAGTTCTTGTAACTG-3′wbjC2R (−)5′-GACAAGCTTCTCACTATACCTTACGCACC-3′wbjD1F (+)5′-CGAGATCTACCATGGAGAAGCTAAAAGTCG-3′wbjD2R (−)5′-GAGAATTCGGATCCTTGCCATCAACTCC-3′wzy11F (+)5′-GTTATGGTTCGATCTATATGG-3′cap5E1F (+)5′-GCAGAGCTCCATGGTCGATGACAAAATTTTAT-3′cap5E2R (−)5′-TGGGATCCGGTACCTCTCCTATCTCATTGAAGC-3′T7 (+)5′-TAATACGACTCACTATAGGG-3′gent1F (+)5′-GTTAGGTGGCTCAAGTATGG-3′gent2R (−)5′-AGATCACATAAGCACCAAGC-3′KKI (+)5′-ACAATCTAGAGCCAGATACGTATTTCTTGG-3′KK2 (−)5′-ACAAGAATTCCATTTCCTCCAAGTATTTCG-3′5E-F (+)5′-GGAGGCTAGCATGTTCGATGACAAAATT-3′5E-R (−)5′-CTCTCGAGTCTCATTGAAGCTTTATAAT-3′5F-F (−)5′-GATAGGCTAGCTTGACGTTGAAT-3′5F-R (−)5′-TCCATGCGCTCGAGCTCCAAGA-3′12402F1 (+)5′-GCATGAGCTCAAGGTGGCGAAGTATT-3′12402R2 (−)5′-TGGAGTTCCTTCAATAGCACGCTTTA-3′12402F2 (+)5′-CAGTCTAGACTCGATCGAACATTGCC-3′12402R2 (−)5′-TTATCTAGACAATCGCTATCCTCATC-3′2-a Forward and reverse primers are represented by plus and minus signs, respectively.2-b The underlined sequences represent the restriction sites used to clone the PCR products. Open table in a new tab S. aureus cap5E, capF, and capG genes were PCR-amplified from strain Newman chromosomal DNA with High Fidelity PCR Supermix (Invitrogen). Primers 5E-F and 5E-R were utilized to amplify the cap5E gene. This amplicon was digested withXhoI and NheI and ligated to pET24a+ digested with the same enzymes. The resultant plasmid pKBK50d contained the ribosomal-binding site and the ATG start site of the pET vector in-frame with the entire cap5E gene and C-terminal six-histidine tag. A similar strategy was applied to clone thecap5F gene; it was amplified with primers 5F-F and 5F-R and cloned into the pET24a+ vector, creating plasmid pET5F1.1. Thecap5G gene was amplified with KK1 and KK2. The 1.18-kb amplicon was digested with XbaI and EcoRI and ligated to appropriately digested pET24c+, yielding plasmid pKBK6a with a C-terminal histidine tag. All of the plasmids were confirmed by nucleotide sequencing of the PCR-amplified genes. To construct the plasmids containing a nonpolar gene disruption ofP. aeruginosa wbjB, the PCR product described above was ligated into SmaI-cut pEX100T, taking advantage of the blunt-end PCR product made by Pwo polymerase. ThewbjB-containing plasmid pKOB1 was subsequently cut withKpnI, and the aacC1 gene (gentamicin resistance cassette) from pUCGm was ligated into pKOB1 to yield thewbjB knockout plasmid pKOB2. To create a plasmid for S. aureus cap5G gene disruption, subclone pJCL69 (containing the end of cap5E through the start of cap5I) was digested with BclI, releasing a 294-bp fragment 50 bp downstream of the ATG start site ofcap5G yielding pJCL69-2. The ermB gene from pERMB was ligated into the BclI site of pJCL69-2 to create pJCL69-2ermB. A 4402-bp XbaI-BamHI fragment from pJCL69-2ermB was ligated to digested pCL10 to yield pKOR1. To create a knockout plasmid for use in cap5F allelic replacement, a modified pCL10 plasmid containing ermB in theBamHI site was constructed (pAP1.2EX). A 1.6-kb region of the cap5 gene locus (containing 450 bp of cap5Fgene and 1124 bp of the cap5G gene) was PCR-amplified using primers 12402F2 and 12402R2, digested with XbaI, and ligated to XbaI-digested pAP1.2EX to form pKOR3. A 1941-bp region of the cap5 locus containing 385 bp of cap5D, the complete open reading frame (1026 bp) of cap5E gene, and 530 bp of cap5F was PCR-amplified using primers 12402F1 and 12402R1. This amplicon was digested with SacI andEcoRV and ligated to similarly digested pKOR3 to yield the 11.32-kb plasmid pKOR4. pFuc11 and pFuc21 plasmid DNA was used to amplify the genes for the complementation plasmids. PCR was carried out with T7 forward primer and the corresponding reverse primers used to get the expression plasmids. The XbaI site in the T7 promoter sequence was used to ligate the PCR product into pUCP27 digested withXbaI/EcoRI for wbjB orXbaI/KpnI in the case of cap5E. Thus, we could take advantage of the ribosomal-binding site of the pET vectors and achieve complementation with the N-terminally histidine-tagged fusion proteins. The resulting plasmids pPAB27 and pSAE27 were confirmed by DNA sequencing. S. aureusplasmid pKOR2 was constructed by ligating a 2.2-kbBamHI-XbaI fragment from pJCL43 containing thecap5G gene along with 920 bp of upstream and 144 bp of downstream flanking sequences into the E. coli/S. aureus shuttle vector pLI50. Expressions were carried out at 37 °C using terrific broth (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), supplemented with 50 μg/ml kanamycin for WbjB and WbjC, with 25 μg/ml kanamycin for Cap5E, Cap5F, or Cap5G, or with 250 μg/ml ampicillin for WbjD. The cultures were grown to an A 600 nm of 0.6, and expression was induced with isopropyl-1-thio-β-d-galactopyranoside at a final concentration of 1 mm. WbjB ,WbjD, Cap5E, and Cap5F expression was carried out for 3 h at 37 °C, WbjC was expressed for 4 h at 37 °C, and Cap5G was expressed for 4 h at 30 °C. The cells were disrupted on ice by ultrasonication. Cell debris and membrane fractions were removed by ultracentrifugation at 300,000 × g. Purifications using nickel chelating columns were performed as recommended by the manufacturers. Purified WbjC and WbjD could be obtained at 200 mm imidazole, whereas WbjB, Cap5E, Cap5F, and Cap5G were eluted from the column at 300 mm imidazole. Dithiothreitol was added to a final concentration of 1 mm, and the proteins were stored at −20 °C after the addition of 40% glycerol. The purity of the enzymes was checked by SDS-PAGE analysis on a 10% gel, and N-terminal protein sequencing was performed on Cap5E, Cap5F, and Cap5G to verify the purified proteins. CE analysis was performed with a P/ACE MDQ Glycoprotein system with UV detection (Beckman Coulter, Fullerton, CA). The samples were separated at 22 kV in 25 mm borate buffer (pH 9.5) at 25 °C. Typically, the reactions contained 0.5 mm UDP-d-GlcNAc, 0.1 mm NADP+, and an excess of hydride donor (NADH or NADPH). For CE-MS analysis, the UDP-d-GlcNAc concentration was 0.1 or 0.5 mm. The standard reaction buffer was 20 mm Tris-HCl at pH 8.0 supplemented with 10 mm MgCl2. The enzymes (∼1 μg each) were added, and the reaction mixtures were incubated at 37 °C for 60 min before being analyzed by CE. The reaction cascades were investigated as stepwise reactions adding one enzyme at a time (allowing optimal substrate conversion on each reaction step) and as combined reactions adding more than one enzyme to start the reaction. The crystal model 310 CE instrument (ATI Unicam, Boston, MA) was coupled to an API 3000 mass spectrometer (MDS/Sciex, Concord, Canada) via a micro Ionspray interface. A sheath solution (isopropanol:methanol, 2:1) was delivered at a flow rate of 1 nl/min to a low dead volume tee (internal diameter, 250 μm; Chromatographic Specialties, Brockville, Canada). The separation was achieved using a 90-cm bare fused silica capillary with a buffer of 30 mm morpholine/formic acid in deionized water, pH 9.0, with 5% methanol. For positive detection mode, a 10 mm ammonium acetate buffer, pH 9.0, containing 5% methanol was used. A separation voltage of 30 kV was typicall" @default.
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• W2011574741 date "2003-02-01" @default.
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• W2011574741 title "Three Highly Conserved Proteins Catalyze the Conversion of UDP-N-acetyl-d-glucosamine to Precursors for the Biosynthesis of O Antigen in Pseudomonas aeruginosaO11 and Capsule in Staphylococcus aureus Type 5" @default.
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• W2011574741 doi "https://doi.org/10.1074/jbc.m203867200" @default.
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