Kahler C M, Carlson R W, Rahman M M, Martin L E, Stephens D S
Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
J Bacteriol. 1996 Mar;178(5):1265-73. doi: 10.1128/jb.178.5.1265-1273.1996.
A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.
在一个Tn916（tetM）突变体文库中，通过与单克隆抗体3F11失去反应性，鉴定出了B群脑膜炎奈瑟菌菌株NMB（免疫型L3,7,9）的脂寡糖（LOS）突变体。该单克隆抗体识别野生型LOS结构中乳糖-N-新四糖部分的末端Galβ1→4GlcNAc表位。这个被命名为559的突变体被发现表达一种3.0 kDa的截短型LOS。Southern杂交和PCR分析表明，在突变体559的染色体上有一个单一完整的Tn916插入（I类）。通过野生型亲本的转化，证实了LOS表型与Tn916插入之间的连锁关系。对转座位点周围区域的核苷酸序列分析揭示了一个1065 bp的开放阅读框（ORF）。对GenBank/EMBL数据库进行同源性搜索发现，这个ORF的氨基酸序列与鼠伤寒沙门氏菌的α1,2 N-乙酰葡糖胺转移酶（RfaK）有46.8%的相似性和21.2%的同一性。对突变体559产生的LOS进行糖基组成和连接分析表明，连接在庚糖I（HepI）上的乳糖-N-新四糖基团以及连接在亲本LOS内核中庚糖II（HepII）上的N-乙酰葡糖胺和葡萄糖残基都不存在。这些分析还表明，亲本和突变体LOS分子中的HepII残基都被磷酸化了，推测是由磷酸乙醇胺取代基磷酸化的。将非极性和极性抗生素抗性盒插入亲本rfaK基因导致表达出与突变体559产生的LOS具有相同迁移率的LOS。这个结果表明，559 LOS无法添加乳糖-N-新四糖基团不是由于对rfaK下游基因的极性效应。我们的数据表明，我们已经鉴定出了负责将N-乙酰葡糖胺添加到HepII上的脑膜炎球菌α1,2 N-乙酰葡糖胺转移酶。我们提出，突变体559的LOS中HepI缺乏α链延伸可能是由于LOS内核不完全生物合成所施加的结构限制。
