Amor P A, Yethon J A, Monteiro M A, Whitfield C
Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1.
J Bacteriol. 1999 Feb;181(3):772-80. doi: 10.1128/JB.181.3.772-780.1999.
Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40LPS backbone structure is composed of a trisaccharide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an L-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145-161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40LPS form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40LPS reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that orf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3 also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the L-serine substitution of GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the L-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with L-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.
大肠杆菌O8:K40在其表面共表达两种不同的脂多糖（LPS）结构。O8多糖是一种具有三糖重复单元的甘露糖同聚物，通过ABC-2转运依赖途径合成。K40 LPS主链结构由N-乙酰葡糖胺（GlcNAc）和葡糖醛酸（GlcA）的三糖重复单元组成，并且有一个不常见的取代基，即连接在葡糖醛酸上的L-丝氨酸部分。先前已克隆并测序了负责K40多糖合成的基因簇，发现其包含六个开放阅读框（ORF）（P.A. Amor和C. Whitfield，《分子微生物学》26:145 - 161，1997）。在此，我们证明orf1的插入失活导致一种半粗糙（SR）-K40 LPS形式的积累，该形式在Western免疫印迹中与特异性多克隆血清保持反应性。对SR-K40 LPS的结构和组成分析表明，它由连接到脂质A核心的单个K40重复单元组成。K40多糖缺乏聚合表明orf1编码K40聚合酶（Wzy），并且K40多糖的组装通过Wzy依赖途径发生（与O8多糖相反）。orf3的失活也导致一种SR-LPS形式的积累，该形式在Western免疫印迹中不与特异性多克隆K40血清反应。对这种SR-LPS的甲基化连接分析和快原子轰击质谱显示，K40多糖的生物学重复单元是GlcNAc-GlcA-GlcNAc。此外，该结构缺乏GlcA的L-丝氨酸取代。这些结果表明：（i）orf3编码负责将L-丝氨酸残基添加到K40主链的酶；（ii）用L-丝氨酸对单个K40重复单元进行取代对于它们被Wzy识别并聚合成K40多糖至关重要。
