SELECTIVE SYNTHESIS AND LABELING OF THE POLYSIALIC ACID CAPSULE IN ESCHERICHIA-COLI-K1 STRAINS WITH MUTATIONS IN NANA AND NEUB
SELECTIVE SYNTHESIS AND LABELING OF THE POLYSIALIC ACID CAPSULE IN ESCHERICHIA-COLI-K1 STRAINS WITH MUTATIONS IN NANA AND NEUB
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DOI：
10.1128/jb.174.19.6191-6197.1992
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发表时间：
1992-10-01
影响因子：
3.2
通讯作者：
VIMR, ER
中科院分区：
文献类型：
--
作者：
VIMR, ER
在大肠杆菌K1中合成聚唾液酸胶囊所需的酶是编码多基因KPS簇的区域2 neu基因。为了便于分析衣壳的合成和转位，通过转导构建了一株nana和neub突变分别影响唾液酸降解和合成的大肠杆菌K1菌株。突变体的无囊表型在体内通过外源唾液酸的加入而得到纠正。通过阻止唾液酸降解，NaNA突变允许细胞内代谢物积累，而Neub突变防止内源唾液酸池稀释，并允许通过在生长介质中添加外源唾液酸来实验控制胶囊合成。用噬菌体K1F吸附试验或感染力试验检测互补作用。在添加唾液酸后的2min内观察到聚唾液酸的转运，表明唾液酸在体内的转运、活化以及多糖聚合和转运到细胞表面的速度很快。氯霉素不抑制噬菌体对噬菌体的吸附，说明37℃下合成或转位多唾液酸不需要从头合成蛋白质。外源放射性标记唾液酸被特异性地掺入到胶囊多糖中。凝胶渗透层析和唾液酸基聚合物对K1F内切N-酰基神经氨酸酶的敏感性证实了标记囊膜材料的聚合性。实验上操控囊膜表达的能力为研究聚唾液酸的合成和膜转位机制提供了新的途径。
The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded hy region 2 neu genes of the multigenic kps cluster. To facilitate analysis of capsule synthesis and translocation, an E. coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction. The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid. By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium. Complementation was detected by bacteriophage K1F adsorption or infectivity assays. Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface. Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37-degrees-C. Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide. The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase. The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms.