Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution
Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution
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DOI：
10.1111/j.1432-1033.1997.00716.x
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发表时间：
1997-07-15
期刊：
影响因子：
--
通讯作者：
Brade, H
中科院分区：
文献类型：
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作者：
Brabetz, W;MullerLoennies, S;Brade, H
通过等位基因置换诱变使大肠杆菌W3110的染色体基因rfaC和rfaF失活，以产生缺乏两种庚糖基转移酶的限定菌株，所述庚糖基转移酶在脂多糖（LPS）生物合成中催化前两个L-甘油-D-甘露-庚糖（Hep）残基转移至3-脱氧-D-甘露-2-辛酮糖酸（Kdo）。分离出突变体的LPS，并通过分离的脱酰基低聚糖磷酸盐的组成分析和核磁共振光谱研究其化学结构。基本结构是四糖α-Kdo-（2->4）-α-Kdo-（2->6）-β-D-GlcN 4P-（1->6）-α-D-GlcN 1 P，其在LPS中在Kdo II的O 7位被非化学计量量的磷酸2-氨基乙醇取代。2-氨基乙醇被裂解的LPS通过连续肼解和KOH处理的脱酰化过程中，此外，磷酸盐迁移从O 7到O 8的Kdo II发生。因此，可以分离寡糖α-Kdo 7 P-（2->4）-α-Kdo-（2->6）-β-D-GlcN 4 P-（1->6）-α-D-GlcN 1 P和α-Kdo 8 P-（2->4）-α-Kdo-（2->6）-β-D-GlcN 4 P-（1->6）-α-D-GlcN 1 P。KOH处理的两个三磷酸和真正的甲基3-脱氧-D-甘露-octulopyranoside 7-（2-乙酰氨基乙基磷酸）证明，磷酸盐迁移只发生时，磷酸基团被2-氨基乙醇取代。用质粒编码的rfaC和rfaF基因进行的互补研究表明，突变菌株可与LPS特异性抗体组合用于克隆和表征使LPS内核区域的I（do）残基糖基化的庚糖基转移酶。
The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic-replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L-glycero-D-manno-heptose (Hep) residues to 3-deoxy-D-manno-2-octulosonic acid (Kdo). The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates. The basic structure was a tetrasaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D-GlcN1P which in LPS was substituted at position O7 of Kdo II by 2-aminoethanol phosphate in non-stoichiometric amounts. 2-Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from O7 to O8 of Kdo II occurred. Thus, the oligosaccharides alpha-Kdo7P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D-GlcN1P and alpha-Kdo8P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D-GlcN1P could be isolated. KOH treatment of the two trisphosphates and authentic methyl 3-deoxy-D-manno-octulopyranoside 7-(2-acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2-aminoethanol. Complementation studies with plasmid-encoded, rfaC and rfaF genes revealed that the mutant strain can be used in combination with LPS-specific antibodies for the cloning and characterization of heptosytransferases which glycosylate I(do residues of the inner core region of LPS.