Beutin Lothar, Strauch Eckhard, Zimmermann Sonja, Kaulfuss Stefan, Schaudinn Christoph, Männel Andrea, Gelderblom Hans R
Department of Biological Safety, Robert Koch-Institut, Berlin, D-13353, Germany.
BMC Microbiol. 2005 Jan 24;5:4. doi: 10.1186/1471-2180-5-4.
Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.
The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.
The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strains which were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigh are functionally expressed and coassembled in the same flagellar filament in E. coli.
O抗原（脂多糖）和H抗原（鞭毛）血清型鉴定是一种广泛用于鉴定大肠杆菌致病菌株和克隆株的方法。目前，已描述了大肠杆菌的176种O抗原和53种H抗原，它们在菌株中以不同组合出现。鞭毛抗原H4广泛存在于不同O血清型和致病型的大肠杆菌菌株中，我们通过聚合酶链反应（PCR）、核苷酸测序和表达研究，对编码H4的鞭毛蛋白基因（fliC基因）之间的遗传关系进行了研究。
测定了大肠杆菌参考菌株U9-41（O2:K1:H4）和P12b（O15:H17）中fliC基因的完整核苷酸序列，发现二者编码序列的同源性为99.3%（1050个核苷酸中的1043个）。开发了一种用于fliC-H4菌株分型的PCR/限制性片段长度多态性（RFLP）方法，并对88株与H4抗血清发生反应的大肠杆菌菌株进行了研究。根据血清凝集效价、fliC-PCR基因分型和参考数据，选择了6株大肠杆菌菌株，对其完整fliC基因进行核苷酸测序，结果显示其氨基酸水平的同源性为96.6%至100%。在编码H48鞭毛型的大肠杆菌K-12菌株JM109中，研究了来自菌株U9-41的fliC-H4和我们的菌株P12b（一种表达H4的变异型）所编码鞭毛蛋白的功能表达。携带fliC重组质粒的JM109菌株与H4和H48特异性抗血清均发生反应，而JM109仅与H48抗血清发生反应。通过免疫电子显微镜，我们能够证明携带fliC-H4重组质粒的菌株所产生的鞭毛由H48和H4鞭毛蛋白组成，它们共同组装成功能性鞭毛。
鞭毛血清型H4由存在于不同血清学类型的大肠杆菌菌株中的密切相关的fliC基因编码，这些菌株是在不同时期和地理位置分离得到的。我们的表达研究首次表明，不同分子量的鞭毛蛋白在大肠杆菌中能够进行功能表达并共同组装在同一鞭毛丝中。
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