Kröncke K D, Boulnois G, Roberts I, Bitter-Suermann D, Golecki J R, Jann B, Jann K
Max-Planck-Institut für Immunbiologie, Freiburg, Federal Republic of Germany.
J Bacteriol. 1990 Feb;172(2):1085-91. doi: 10.1128/jb.172.2.1085-1091.1990.
The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.
荚膜K5多糖是大肠杆菌II型荚膜抗原的代表，此前已被克隆，并且已确定了负责聚合和表面表达的三个基因区域（I.S.罗伯茨、R.芒福德、R.霍奇、K.B.扬和G.J.布尔诺瓦，《细菌学杂志》170:1305 - 1310，1988年）。在本报告中，我们描述了表达K5抗原的重组细菌以及区域1或区域3基因功能有缺陷的突变体的免疫电子显微镜分析，以及K5荚膜多糖的生化分析。虽然K5克隆在其约25%的群体中表达K5多糖形成发育良好的荚膜，但在整装标本以及表达突变体的超薄切片中未观察到荚膜。对区域3突变体切片的免疫金标记显示细胞质中有荚膜K5多糖。对于区域1突变体，荚膜多糖似乎与细胞膜相关，并且与区域3突变体多糖不同，在细菌质壁分离后可在周质中检测到荚膜多糖。用十六烷基三甲基溴化铵从匀浆的突变体中分离多糖。区域1突变体的多糖与从原始K5克隆的荚膜中分离的多糖大小相同，并且两种多糖都被磷脂酸取代。区域3突变体的多糖较小且未被磷脂酸取代。这些结果促使我们推测，基因区域3的产物参与荚膜多糖跨细胞质膜的转运，而区域1指导脂质取代的荚膜多糖通过周质并穿过外膜的运输。
