Identification of H-specific determinants in flagellin of four Escherichia coli strains
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O R I G I N A L PA P E R
June-Nam Seah · Jimmy Kwang
Identification of H-specific determinants in flagellin of four Escherichia coli strains
Received: 30 December 1999 / Revised: 30 March 2000 / Accepted: 6 April 2000 / Published online: 18 May 2000 © Springer-Verlag 2000
Abstract The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, O138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305–331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430–461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171–201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200–260. Key words Escherichia coli · Flagella · Flagellin · fliC · Mapping · Serotype Abbreviations ECRC Escherichia coli Reference Center · CVR Central variable region · GST Glutathione-S-transferase
Introduction In Salmonella and Escherichia species, the flagellum is a complex consisting of a basal body, a hook, and a filament (Macnab 1992). The flagellar filament is a homopolymer
J.N. Seah · J. Kwang (✉) Animal Health Biotechnology, Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 e-mail: [email protected], Tel.: +65-8727473, Fax: +65-8727007
composed of a repeated protein, flagellin (Joys 1988), which in Escherichia coli is encoded by the fliC gene (Kuwajima et al. 1986). Morphologically, flagella of different H-types found in E. coli can be grouped into six morphotypes (A, B, C, D, E and F) according to their morphology as observed by electron microscopy (Lawn et al. 1977). Sequence analysis of the fliC genes for different serological specificities reveals highly conserved N- and C-terminal regions and a central region with considerable variation (Kuwajima et al. 1986; Schoenhals and Whitfield 1993). The molecular weight of flagellin also varies greatly among E. coli H-types (Lawn 1977; Schoenhals and Whitfield 1993). The N- and C-terminal conserved regions of the fliC gene are essential for the polymerization and secretion of flagellin molecules (Homma et al. 1987; Hyman and Trachtenberg 1991; Kanto et al. 1991; Kuwajima et al. 1989). The central variable region (CVR) of flagellin encodes major determinants for different H-types of E. coli (Feng et al. 1990; Kuwajima 1988a; Kwang et al. 1996; Schoenhals and Whitfield 199
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