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• W1997524999 abstract "The lipooligosaccharide (LOS) ofHaemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgBaffected only the sialylated glycoforms containingN-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, andN-acetylneuraminic acid. Chromosomal complementation of thesiaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lackingN-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant insiaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in thesiaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction inN-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases. The lipooligosaccharide (LOS) ofHaemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgBaffected only the sialylated glycoforms containingN-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, andN-acetylneuraminic acid. Chromosomal complementation of thesiaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lackingN-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant insiaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in thesiaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction inN-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases. Haemophilus influenzae frequently colonizes the human nasopharynx. Up to 80% of the population harbor this organism as part of their normal flora (1.Turk D.C. Sell S.H. Wright P.F. Haemophilus influenzae: Epidemiology, Immunology, and Prevention of Disease. Elsevier Science Publishing Co., Inc., New York1982: 3-9Google Scholar). Although normally an innocuous inhabitant of the upper respiratory tract, H. influenzae is an opportunistic pathogen. The diseases caused by the organism can be ordered in two groups based on the presence or absence of a capsule. Encapsulated or typeable organisms, which range from capsule types a–f, can cause systemic infections such as bacteremia, septicemia, and bacterial meningitis (1.Turk D.C. Sell S.H. Wright P.F. Haemophilus influenzae: Epidemiology, Immunology, and Prevention of Disease. Elsevier Science Publishing Co., Inc., New York1982: 3-9Google Scholar, 2.Pittman M. J. Exp. Med. 1931; 53: 471-492Crossref PubMed Scopus (290) Google Scholar). Of the various encapsulated types,H. influenzae type b has been associated most often with pathogenesis (2.Pittman M. J. Exp. Med. 1931; 53: 471-492Crossref PubMed Scopus (290) Google Scholar). The nonencapsulated or nontypeable (NTHi) 1The abbreviations used are: NTHinontypeableH. influenzaeLOSlipooligosaccharidePEAphosphoethanolamineORFopen reading frameELISAenzyme-linked immunosorbent assayO-LOSO-deacylated lipooligosaccharideMALDI-MSmatrix-assisted laser desorption ionization mass spectrometryHexhexoseHexNAcN-acetylhexosamineKdo2-keto-3-deoxy-d-manno-octulosonic acidHepl-glycero-d-manno-heptose ord-glycero-d-manno-heptoseTOFtime-of-flight mass analyzer[M-H]−deprotonated molecular ion.1The abbreviations used are: NTHinontypeableH. influenzaeLOSlipooligosaccharidePEAphosphoethanolamineORFopen reading frameELISAenzyme-linked immunosorbent assayO-LOSO-deacylated lipooligosaccharideMALDI-MSmatrix-assisted laser desorption ionization mass spectrometryHexhexoseHexNAcN-acetylhexosamineKdo2-keto-3-deoxy-d-manno-octulosonic acidHepl-glycero-d-manno-heptose ord-glycero-d-manno-heptoseTOFtime-of-flight mass analyzer[M-H]−deprotonated molecular ion. strains of H. influenzae cause more localized infections, such as chronic bronchitis or otitis media, and rarely cause systemic infections (3.Murphy T.F. Apicella M.A. Rev. Infect. Dis. 1987; 9: 1-15Crossref PubMed Scopus (253) Google Scholar,4.Murphy T.F. Semin. Respir. Infect. 2000; 15: 41-51Crossref PubMed Scopus (47) Google Scholar). nontypeableH. influenzae lipooligosaccharide phosphoethanolamine open reading frame enzyme-linked immunosorbent assay O-deacylated lipooligosaccharide matrix-assisted laser desorption ionization mass spectrometry hexose N-acetylhexosamine 2-keto-3-deoxy-d-manno-octulosonic acid l-glycero-d-manno-heptose ord-glycero-d-manno-heptose time-of-flight mass analyzer deprotonated molecular ion. nontypeableH. influenzae lipooligosaccharide phosphoethanolamine open reading frame enzyme-linked immunosorbent assay O-deacylated lipooligosaccharide matrix-assisted laser desorption ionization mass spectrometry hexose N-acetylhexosamine 2-keto-3-deoxy-d-manno-octulosonic acid l-glycero-d-manno-heptose ord-glycero-d-manno-heptose time-of-flight mass analyzer deprotonated molecular ion. There are a number of virulence factors associated with both H. influenzae type b and NTHi that contribute to their pathogenesis, one of these being the lipooligosaccharide (LOS) (5.Kimura A. Hansen E.J. Infect. Immun. 1986; 51: 69-79Crossref PubMed Google Scholar, 6.Kimura A. Patrick C.C. Miller E.E. Cope L.D. McCracken Jr., G.H. Hansen E.J. Infect. Immun. 1987; 55: 1979-1986Crossref PubMed Google Scholar, 7.Zwahlen A. Rubin L.G. Moxon E.R. Microb. Pathog. 1986; 1: 465-473Crossref PubMed Scopus (78) Google Scholar, 8.Cope L.D. Yogev R. Mertsola J. Argyle J.C. McCracken Jr., G.H. Hansen E.J. Infect. Immun. 1990; 58: 2343-2351Crossref PubMed Google Scholar, 9.Swords W.E. Buscher B.A. Ver Steeg Ii K. Preston A. Nichols W.A. Weiser J.N. Gibson B.W. Apicella M.A. Mol. Microbiol. 2000; 37: 13-27Crossref PubMed Scopus (256) Google Scholar). LOS is a complex glycolipid containing three main regions: lipid A, core, and a variable branched region (10.Preston A. Mandrell R.E. Gibson B.W. Apicella M.A. Crit. Rev. Microbiol. 1996; 22: 139-180Crossref PubMed Scopus (240) Google Scholar). The core region is a conserved structure containing a phosphorylated Kdo residue linked to three heptose residues, whereas the variable branched region contains a heterogeneous mix of hexoses and N-acetylhexosamines as well as other factors, such as phosphoethanolamine (PEA), phosphorylcholine, and NeuAc (11.Flesher A.R. Insel R.A. J. Infect. Dis. 1978; 138: 719-730Crossref PubMed Scopus (60) Google Scholar, 12.Inzana T.J. Seifert Jr., W.E. Williams R.P. Infect. Immun. 1985; 48: 324-330Crossref PubMed Google Scholar, 13.Parr Jr., T.R. Bryan L.E. Can. J. Microbiol. 1984; 30: 1184-1187Crossref PubMed Scopus (7) Google Scholar, 14.Zamze S.E. Moxon E.R. J. Gen. Microbiol. 1987; 133: 1443-1451PubMed Google Scholar, 15.Phillips N.J. Apicella M.A. Griffiss J.M. Gibson B.W. Biochemistry. 1993; 32: 2003-2012Crossref PubMed Scopus (81) Google Scholar, 16.Risberg A. Masoud H. Martin A. Richards J.C. Moxon E.R. Schweda E.K. Eur. J. Biochem. 1999; 261: 171-180Crossref PubMed Scopus (101) Google Scholar). LOS differs from its enterobacterial counterpart, lipopolysaccharide, in that the variable branched region or O-antigen is a nonrepeating unit (10.Preston A. Mandrell R.E. Gibson B.W. Apicella M.A. Crit. Rev. Microbiol. 1996; 22: 139-180Crossref PubMed Scopus (240) Google Scholar). A great deal of work has been undertaken to understand the biosynthesis and role of LOS in pathogenesis (10.Preston A. Mandrell R.E. Gibson B.W. Apicella M.A. Crit. Rev. Microbiol. 1996; 22: 139-180Crossref PubMed Scopus (240) Google Scholar). H. influenzae LOS is very heterogeneous and contains a number of phase-varying epitopes (17.Inzana T.J. J. Infect. Dis. 1983; 148: 492-499Crossref PubMed Scopus (109) Google Scholar, 18.Patrick C.C. Kimura A. Jackson M.A. Hermanstorfer L. Hood A. McCracken Jr., G.H. Hansen E.J. Infect. Immun. 1987; 55: 2902-2911Crossref PubMed Google Scholar). Phase variation is known at least in part to occur through a process of slipped-strand mispairing (19.Weiser J.N. Love J.M. Moxon E.R. Cell. 1989; 59: 657-665Abstract Full Text PDF PubMed Scopus (240) Google Scholar). Three well characterized loci involved in LOS expression and phase variation, designated lic1, lic2, andlic3, phase-vary through this mechanism (20.Maskell D.J. Szabo M.J. Butler P.D. Williams A.E. Moxon E.R. Mol. Microbiol. 1991; 5: 1013-1022Crossref PubMed Scopus (55) Google Scholar, 21.Weiser J.N. Lindberg A.A. Manning E.J. Hansen E.J. Moxon E.R. Infect. Immun. 1989; 57: 3045-3052Crossref PubMed Google Scholar, 22.Weiser J.N. Maskell D.J. Butler P.D. Lindberg A.A. Moxon E.R. J. Bacteriol. 1990; 172: 3304-3309Crossref PubMed Google Scholar). Phase variation may play a role for the bacterium in the evasion of the host immune response. LOS structures have also been found to mimic human blood group antigens, such as the Pk antigen and paragloboside (23.Mandrell R.E. McLaughlin R. Aba Kwaik Y. Lesse A. Yamasaki R. Gibson B. Spinola S.M. Apicella M.A. Infect. Immun. 1992; 60: 1322-1328Crossref PubMed Google Scholar). This may be another method for bacterial immune evasion. The lsg (lipooligosaccharidesynthesis genes) locus is another region involved in LOS biosynthesis (24.Spinola S.M. Kwaik Y.A. Lesse A.J. Campagnari A.A. Apicella M.A. Infect. Immun. 1990; 58: 1558-1564Crossref PubMed Google Scholar). Seven genes are in the locus, six of which have identity to various glycosyltransferases and one that acts as a regulator (25.Phillips N.J. Miller T.J. Engstrom J.J. Melaugh W. McLaughlin R. Apicella M.A. Gibson B.W. J. Biol. Chem. 2000; 275: 4747-4758Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This locus is not controlled by the slipped strand mispairing mechanism. Through studies expressing chimericHaemophilus structures in Escherichia colilipopolysaccharide, we know that this locus is involved in the expression of a terminal N-acetyllactosamine structure (25.Phillips N.J. Miller T.J. Engstrom J.J. Melaugh W. McLaughlin R. Apicella M.A. Gibson B.W. J. Biol. Chem. 2000; 275: 4747-4758Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). One of the genes in this locus, lsgB, has homology (27% identity, 46% similarity) to the sialyltransferase in Neisseria meningitidis. In various Neisseria andHaemophilus species, a terminalN-acetyllactosamine structure has been shown to be an acceptor for sialylation (23.Mandrell R.E. McLaughlin R. Aba Kwaik Y. Lesse A. Yamasaki R. Gibson B. Spinola S.M. Apicella M.A. Infect. Immun. 1992; 60: 1322-1328Crossref PubMed Google Scholar, 26.Phillips N.J. McLaughlin R. Miller T.J. Apicella M.A. Gibson B.W. Biochemistry. 1996; 35: 5937-5947Crossref PubMed Scopus (34) Google Scholar, 27.Mandrell R.E. Griffiss J.M. Macher B.A. J. Exp. Med. 1988; 168: 107-126Crossref PubMed Scopus (185) Google Scholar). NeuAc is a constituent of the LOS in about half of the H. influenzae strains tested (23.Mandrell R.E. McLaughlin R. Aba Kwaik Y. Lesse A. Yamasaki R. Gibson B. Spinola S.M. Apicella M.A. Infect. Immun. 1992; 60: 1322-1328Crossref PubMed Google Scholar, 28.Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). Sialylation in H. influenzae has been shown to affect its ability to evade the lytic effects of human serum (28.Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar, 29.Hood D.W. Cox A.D. Gilbert M. Makepeace K. Walsh S. Deadman M.E. Cody A. Martin A. Mansson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Wakarchuk W.W. Mol. Microbiol. 2001; 39: 341-351Crossref PubMed Scopus (109) Google Scholar). Two genes have been identified that are involved in LOS sialylation, siaB and lic3A(28.Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar, 29.Hood D.W. Cox A.D. Gilbert M. Makepeace K. Walsh S. Deadman M.E. Cody A. Martin A. Mansson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Wakarchuk W.W. Mol. Microbiol. 2001; 39: 341-351Crossref PubMed Scopus (109) Google Scholar). siaB is a CMP-NeuAc synthetase, and a mutation in this gene eliminates all sialylation (28.Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). The second gene,lic3A, has been shown to function as an α2–3-sialyltransferase, responsible for sialylating terminal lactose structures. The lic3A gene has about 40% identity tocstII from Campylobacter jejuni (29.Hood D.W. Cox A.D. Gilbert M. Makepeace K. Walsh S. Deadman M.E. Cody A. Martin A. Mansson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Wakarchuk W.W. Mol. Microbiol. 2001; 39: 341-351Crossref PubMed Scopus (109) Google Scholar). This gene is one of two sialyltransferases identified in this organism (30.Gilbert M. Brisson J.R. Karwaski M.F. Michniewicz J. Cunningham A.M. Wu Y. Young N.M. Wakarchuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). A mutation in lic3A in one strain of H. influenzaestill contained sialylated glycoforms, indicating the possibility of a second sialyltransferase in this organism (29.Hood D.W. Cox A.D. Gilbert M. Makepeace K. Walsh S. Deadman M.E. Cody A. Martin A. Mansson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Wakarchuk W.W. Mol. Microbiol. 2001; 39: 341-351Crossref PubMed Scopus (109) Google Scholar). H. influenzae contains a homologue to a sialyltransferase fromHaemophilus ducreyi, which is designated as HI0871 in theH. influenzae Rd genome data base (31.Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D.J.S. Shirley R. Liu L. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Fuhrmann J.L. Geoghagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4649) Google Scholar). In a study looking at a number of genes from H. influenzae and their possible role in LOS biosynthesis, no function for this gene (designatedorfY) was found (32.Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (146) Google Scholar). This gene, which we callsiaA, was studied for its possible role in LOS sialylation. We report evidence that siaA is a sialyltransferase inH. influenzae and that lsgB is required for the biosynthesis of a third, distinct sialylated glycoform, and our evidence strongly suggests that LsgB is the third sialyltransferase. All bacterial strains and plasmids used in this study are listed in Table I. Parental strains 2019, A2, and their derivatives were grown on brain heart infusion agar (Difco) supplemented with 10 μg/ml β-nicotinamide adenine dinucleotide (Sigma) and 10 μg/ml hemin (ICN Biochemicals) at 37 °C. When appropriate, 15 μg/ml ribostamycin (Sigma) (a kanamycin analogue), 1 μg/ml chloramphenicol, 15 μg/ml spectinomycin, and 20 μg/ml NeuAc (Sigma) were added to the media.Table IBacterial strains and vectorsStrainsGenotypeSource or referenceE. coliDH5αF-f80dlacZDM15 D(lacZYA-argF)U169 deoRrecA1 endA1 hsdR17(rK−, mK+) phoA supE44 λ-thi-1gyrA96relA1.InvitrogenE. coli DH10BF-mcrA D(mrr-hsdRMS-mcrBC) f80dlacZDM15DlacX74 deoR recA1 endA1araD139 D(ara, leu)7697 galUgalK λ-rpsL nupGInvitrogenH. influenzae A2Type b strainRef. 24.Spinola S.M. Kwaik Y.A. Lesse A.J. Campagnari A.A. Apicella M.A. Infect. Immun. 1990; 58: 1558-1564Crossref PubMed Google ScholarH. influenzae 2019Non-typeable strainRef. 57.Campagnari A.A. Gupta M.R. Dudas K.C. Murphy T.F. Apicella M.A. Infect. Immun. 1987; 55: 882-887Crossref PubMed Google ScholarH. influenzae A2STFsiaA −This studyH. influenzaeA2STFC.P4siaA −, plus a functionalsiaAThis studyH. influenzaeA2STFIRAsiaA −This studyH. influenzae A2SBsiaB −This studyH. influenzae 276.4lsgE −Ref.26.Phillips N.J. McLaughlin R. Miller T.J. Apicella M.A. Gibson B.W. Biochemistry. 1996; 35: 5937-5947Crossref PubMed Scopus (34) Google ScholarH. influenzae 276.4STFlsgE −,siaA −This studyH. influenzaeA2L3Alic3A −This studyH. influenzae A2STFL3AsiaA −,lic3A −This studyH. influenzaeA2lsgBlsgB −This studyH. influenzae A2STFL3AlsgBsiaA −,lic3A −, lsgB −This studyPlasmidsSelection marker and descriptionSource or referencepCR2.1Ampicillin, kanamycin, TA cloning vectorInvitrogenpBluescript KS II−Ampicillin, cloning vectorStratagenepHS89–21Ampicillin, kanamycin,siaA cloned into pCR2.1This studypBSL15Ampicillin, kanamycin, used as a backbone for pABR3 and pHiCM1Ref. 58.Alexeyev M.F. BioTechniques. 1995; 18 (54, 56): 52PubMed Google ScholarpBSL86Ampicillin, kanamycin, source of the kanamycin cassette for pHS89–106K6Ref. 58.Alexeyev M.F. BioTechniques. 1995; 18 (54, 56): 52PubMed Google ScholarpABR3Ampicillin, spectinomycin, pBSL15 with a spectinomycin cassetteRef. 58.Alexeyev M.F. BioTechniques. 1995; 18 (54, 56): 52PubMed Google ScholarpSiaB2Ampicillin, kanamycin, siaB cloned into pCR2.1This studypSiaB2SpecAmpicillin, kanamycin, spectinomycin, pSiaB2 with a spectinomycin cassette insiaBThis studypHS89–103Ampicillin,siaA from pHS89–21 cloned into pBluescript KS II−This studypHS89–103K6Ampicillin, kanamycin, pHS89–103 with a kanamycin cassette in siaAThis studypTAV1Ampicillin, TA cloning vector constructed from pBluescript KS II−Ref. 59.Borovkov A.Y. Rivkin M.I. BioTechniques. 1997; 22: 812-814Crossref PubMed Scopus (87) Google ScholarpHiCM1Ampicillin, chloramphenicol, pBSL15 with chloramphenicol cassetteThis studypIRAAmpicillin, intergenic region from NTHi 2019 cloned into pTAV1This studypIRACMAmpicillin, chloramphenicol, pIRA with chloramphenicol cassetteThis studypCMRAmpicillin, kanamycin, chloramphenicol, source of chloramphenicol cassetteRef.35.Whitby P.W. Morton D.J. Stull T.L. FEMS Microbiol. Lett. 1998; 158: 57-60Crossref PubMed Google Scholarp0352EXAmpicillin, kanamycin, lic3A gene cloned into pCR2.1This studyp0352EXCMAmpicillin, kanamycin, chloramphenicol, p0353EXCM with a chloramphenicol cassette inlic3AThis studyPGEMLOS2Ampicillin,lsgA, lsgB, lsgC, and lsgDcloned into pGEM3Zf+This studypRSM1775Ampicillin, kanamycin, chloramphenicol, source of a nonpolar chloramphenicol cassetteThis studypGEMLOSABCDAmpicillin, lsgA,B, C, D cloned into pGEM3zf+This studypGEMLOS2ABCDermAmpicillin, erythromycin, pGEMLOSABCD with an erythromycin cassette in lsgBThis studypSAIRCMAmpicillin, kanamycin, chloramphenicol, complementation construct containing the intergenic sequence, chloramphenicol gene, andsiaAThis study Open table in a new tab Chromosomal DNA was isolated using standard protocols. Restriction enzymes were purchased from either New England Biolabs or Promega. Polymerase chain reactions (PCRs) were performed with either Taq DNA Polymerase (Roche Molecular Biochemicals) or the Expand Long Template kit (Roche Molecular Biochemicals). All plasmid constructs were maintained in either E. coli DH5α or DH10B (Invitrogen). Gel purification was performed with SeaPlaque GTG-agarose (BioWittaker Molecular Applications) using standard protocols. DNA was digested to completion with the appropriate restriction enzymes, fractionated in 0.7% agarose gels, and transferred to Hybond-N nylon membranes (Amersham Biosciences). Southern blots were hybridized with probes generated by the random primed digoxygenin DNA labeling kit (Roche Molecular Biochemicals). All blots were processed by following the digoxygenin protocols. Chemiluminescent detection was performed with Kodak XAR-5 or BMR-1 film (Eastman Kodak Co.). DNA was sequenced with an Applied Biosystems automated sequencer using fluorescent terminator dye tags at the DNA Sequencing Facility (University of Iowa). Analysis of the sequence was performed using various programs of the Wisconsin GCG package and the Jellyfish software package developed by Biowire.com (available on the World Wide Web at www.biowire.com). Similarity searches against DNA and protein sequence data bases were performed with the FASTA, BLAST, or BLASTX algorithms. Using the DNA sequence from the H. ducreyi lst gene (33.Bozue J.A. Tullius M.V. Wang J. Gibson B.W. Munson Jr., R.S. J. Biol. Chem. 1999; 274: 4106-4114Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), an open reading frame (ORF) in the H. influenzae Rd data base named HI0871 was identified, which contained 48% identity and 59% similarity over the length of the predicted protein sequence. HI0871 was renamed siaA. Primers were made to amplifysiaA and some flanking DNA from strain 2019 based on the Rd sequence from the TIGR data base (available on the World Wide Web at www.tigr.org/). A 4.3-kb product was amplified using the Expand Long Template PCR kit (Roche Molecular Biochemicals) and primers HSTR8 (5′-CTG CAA AAT ACA GAT AAA GCA ACA CTG GGG-3′) and HSTR9 (5′-CAG CGG CAA GAA ATA TAG GGT TAG AAA AAG C-3′), which was then TA-cloned into pCR2.1 (Invitrogen), forming pHS89-21. This insert was sequenced (accession number AY061634). The 4.3-kb DNA fragment was then subcloned into the EcoRI site of pBluescript KS II−(Stratagene), forming pHS89-103. The insertional mutant ofsiaA was constructed by cloning a PstI-digested kanamycin antibiotic resistance gene from pBSL86 into a uniqueNsiI site in the middle of siaA, forming pHS89-103K6. The orientation of the kanamycin gene was discerned with a restriction digest and was found to be transcribed in the same direction as siaA (data not shown). pHS89-103K6 was linearized with ScaI and transformed into strains A2 and 276.4 (34.Herriott R.M. Meyer E.M. Vogt M. J. Bacteriol. 1970; 101: 517-524Crossref PubMed Google Scholar). Transformants were obtained and analyzed using both PCR with internal siaA primers HSTR1 (5′-GAT GTT ATT TTT ATT TTT GTT A-3′) and HSTR2 (5′-ACT TAG GGT GTA TTT TGG TTC C-3′) and Southern blots (data not shown). Primers SiaBU2 (5′-CGG ACT ATC ATA ACG GGC-3′) and SiaBD2 (5′-CTC AGA ATT CGG GCT TCG-3′) were designed based on the H. influenzae Rd genome. A 1.5-kb DNA fragment was amplified from NTHi 2019 and TA-cloned into pCR2.1, and both DNA strands were sequenced. This new plasmid, called pSiaB2, contained a 675-base pair ORF with 95% identity to HI1279 from H. influenzae Rd. This plasmid was digested with SspI, which cuts at a unique site after nucleotide 276. A spectinomycin resistance cassette gene was digested with SmaI from pABR3 and ligated into the SspI site of pSiaB2, forming pSiaB2Spec. This new construct was digested with NotI to linearize the DNA and then transformed into strain A2 using the MIV method (34.Herriott R.M. Meyer E.M. Vogt M. J. Bacteriol. 1970; 101: 517-524Crossref PubMed Google Scholar). Transformants were obtained and tested using PCR and Southern hybridization to confirm the proper insertion of the spectinomycin cassette in siaB (data not shown). A 3456-bp BamHI-BsbI fragment of H. influenzae A2 DNA containing lsgA,-B, -C, and -D was cloned into pGEM3zf+. A 502-bp (BsrGI-XcmI)region of lsgB was deleted and replaced by an erythromycin cassette. This new plasmid was called pGEMLOS2ABCDerm. This plasmid was digested with NdeI and transformed into strains A2 and A2STFL3A using the MIV method (34.Herriott R.M. Meyer E.M. Vogt M. J. Bacteriol. 1970; 101: 517-524Crossref PubMed Google Scholar). Transformants were obtained and tested using PCR and Southern hybridization to confirm the proper insertion of the erythromycin cassette in lsgB (data not shown). The mutants were designated strains A2lsgB and A2STFL3AlsgB. Primers iraF (5′-AGG GGG ATA AAA CAA AGG-3′) and iraR (5′-GGC AAG TCC CTG TTC AAA-3′) for PCR were designed from the published H. influenzae Rd genome. These primers were used to amplify an intergenic region between bases 794506 and 796038. Amplification resulted in a PCR product of ∼1.6 kb from the genome of NTHi strain 2019. The product was then cloned into the vector pTAV1 via TA cloning. The nucleotide sequence of the cloned fragment was elucidated and compared with sequences included in the genome data base. The resolved consensus sequence was entered into MacVector to identify useful restriction sites. One SphI site was predicted that would cut the cloned region into 711- and 822-base fragments and could be used to linearize pIRA. The presence of the unique SphI site was verified by restriction endonuclease digestion of pIRA. The plasmid pCMR containing a chloramphenicol resistance cassette possessing the consensus uptake sequence for Haemophilus transformation was kindly provided by Dr. Terrence Stull (35.Whitby P.W. Morton D.J. Stull T.L. FEMS Microbiol. Lett. 1998; 158: 57-60Crossref PubMed Google Scholar). To obtain the necessary SphI sites, the chloramphenicol resistance cassette was excised from pCMR using PstI, gel-purified, and then ligated into the vector pBSL15 that had been previously cut with PstI. The resulting plasmid was named pHiCM1. Finally, the chloramphenicol resistance cassette was excised from pHiCM1 by cleavage with SphI, gel-purified, and ligated into the SphI site of pIRA, forming pIRACM. Insertion was confirmed by PCR and by sequencing the cloning junctions. A 2.5-kb EcoRV DNA fragment was excised from pHS89-103, gel-purified, and blunt end-ligated into the SfoI site of pIRACM, forming pSAIRCM. This construct was used as a template for PCR using primers iraR and iraF, and the resulting PCR product was transformed into A2STF using the MIV method (34.Herriott R.M. Meyer E.M. Vogt M. J. Bacteriol. 1970; 101: 517-524Crossref PubMed Google Scholar). Transformants were selected for with both kanamycin and chloramphenicol. Verification that both the full-length and the mutant forms of siaA were present on the chromosome was performed with PCR using primers HSTR1 and HSTR2 and with Southern blots (data not shown). A control strain was constructed in a similar fashion by transforming A2STF with pIRA. The insertion into the chromosome was confirmed with Southern blots, and the resultant strain was named A2STFIRA (data not shown). Primers 0352ELTU1 (5′-ATG TCC AAA AGC AGC CAA CCA AAT AAA CCC-3′) and 0352ELTL1 (5′-CAA CGC CGA AAT CAA CCC AAA TAG AAA GCC-3′) were designed using the H. influenzae Rd genome data base and a 4.6-kb DNA fragment containing the HI0352 ORF (lic3A) was amplified from strain A2 using PCR. The DNA fragment was TA-cloned into pCR2.1 and was named p0352EX. Both DNA strands were sequenced, and a unique SwaI site was found after nucleotide 683 of the 981-nucleotide lic3A sequence. A nonpolar chloramphenicol cassette (pRSM1775), which was constructed in a manner similar to that described by Menard et al. (36.Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (615) Google Scholar) using the chloramphenicol gene (cat) from pACYC184, 2J. A. Bozue and R. S. Munson, Jr., unpublished data. was digested withSmaI and cloned into the SwaI site oflic3A. This construct was named p0352EXCM. The nonpolar cassette contains translation stop codons in all three reading frames upstream from the start codon of cat. Five bases after thecat stop codon, there is a Shine-Dalgarno sequence followed by a start codon, which is in-frame with the remainder of thelic3A gene. The insertion sites of the chloramphenicol cassette were sequenced to ensure proper insertion and orientation. p0352EXCM was linearized by digestion with BamHI and transformed using the MIV method (34.Herriott R.M. Meyer E.M. Vogt M. J. Bacteriol. 1970; 101: 517-524Crossref PubMed Google Scholar) into strains A2 and A2STF, forming strains A2L3A and A2STFL3A respectively. Proper insertion into the chromosome of these strains was confirmed with PCR amplification using primers to the lic3A sequence and Southern hybridization (data not shown). The LOS was prepared by a modification of the Hitchcock and Brown method (37.Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). Organisms were grown on solid media supplemented with 20 μg/ml of NeuAc. The organisms from a single plate were suspended in 2 ml of phosphate-buffered saline buffer to a finalA 65" @default.
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• W1997524999 title "Haemophilus influenzae Type b Strain A2 Has Multiple Sialyltransferases Involved in Lipooligosaccharide Sialylation" @default.
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