Characterization of Two Transposon Mutants from Haemophilus influenzae Type b with Altered Lipooligosaccharide Biosynthesis

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• 作者：Nancy J. Phillips ; Robert McLaughlin ; Theresa J. Miller ; Michael A. Apicella ; and Bradford W. Gibson
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：May 7, 1996
• 年：1996
• 卷：35
• 期：18
• 页码：5937 - 5947
• 全文大小：466K
• 年卷期：v.35,no.18(May 7, 1996)
• ISSN：1520-4995

文摘

Two isogenic mutants of Haemophilus influenzae type b(Hib) strain A2 were prepared byrandom m-Tn3(Cm) insertions into the 7.4-kb lsg(lipooligosaccharide synthesis genes) region of HibDNA, which consists of seven complete and one partial open readingframes (orfs). Compared to theparent A2 strain which produces a complex mixture oflipooligosaccharides (LOS), the mutant strains281.25 and 276.4 produced only a few LOS species. The preciselocations of transposon insertions intothe lsg loci of these mutants were determined (base 3546 inorf 4 for strain 281.25 and base 4402 in orf5 for strain 276.4), and the effects of these mutations on LOSbiosynthesis and epitope expression wereevaluated. When the O-deacylated LOS were analyzed bymass spectrometry, both strains containedmajor LOS species of M_r 2601, 2439, and 2277,which consisted of a common heptose trisaccharidecore structure [Hep₃(PEA)Kdo(P)-lipid A,where Hep isL-glycero-D-manno-heptose,Kdo is 3-deoxy-D-manno-octulosonic acid, and PEA is phosphoethanolamine] andfour, three, or two hexoses, respectively.These species represent the smallest components of the wild-typeLOS mixture. The major LOSoligosaccharide obtained from strain 281.25 by mild acid hydrolysis wasdephosphorylated and shownby composition analysis, methylation analysis, mass spectrometry, and2D NMR studies to be a triantennary structure consisting of a heptose trisaccharide core with twoglucose disaccharide branches:Hep1(Glc14Glc13)2Hep1(Glc14Glc14)3Hep1anhydroKdo.Unlike the parentA2 strain, mutant strain 281.25 cannot add galactoses to the branchesof this octasaccharide. Strain 276.4is similarly deficient, except that it can still utilize a minorbiosynthetic pathway leading to the additionof sialyl-N-acetyllactosamine.