Various deletions were introduced into the central region of Escherichia coli flagellin (497 residues) without destroying its ability to form flagellar filaments. The smallest flagellin retained only the N-terminal 193 residues and the C-terminal 117 residues, which are suggested to be the domains essential for filament formation.The motility of flagellated bacteria stems from the rotation of the external flagellar filament. The filament is a helical, cylindrical structure composed of subunits of flagellin associated each other by noncovalent interactions (11,(24)(25)(26). The flagellin of Escherichia coli K-12, encoded by the hag gene, is composed of 497 amino acid residues (17). Genetic studies of Salmonella typhimurium (6,10,28) and analysis of the homology pattern of the amino acid sequences (2,4,12,17,27) suggest that both the N-and C-terminal regions of flagellin are important for filament formation. Previously, I reported that E. coli flagellins with small deletions in the central region still could assemble into filaments (16). Here, I have extended this approach and have constructed small flagellins which can form filaments despite large deletions in the central region.I had previously constructed plasmids pFD2 and pFD3, in which the hag gene suffers a deletion and an insertion of the 8-mer HindIII linker (16). The deletions in the gene correspond to 16 amino acid residues from residue 239 to residue 254 (pFD2) and 20 residues from residue 259 to residue 278 (pFD3) of E. coli K-12 flagellin. These plasmids were linearized with HindIII, digested with exonuclease Bal3l (19), and ligated in the presence of the 18-mer linker DNA 5'-AAGCTTCCCGGGAGATCT-3', which contains restriction sites for Hindlll, SmaI, and BglII (neither pFD2 nor pFD3 contains SmaI and BglII sites). The resulting plasmids were introduced into E. coli C600 hsm hsr hag::TnJO, which is nonmotile because of the hag mutation (16). From the transformants, I selected eight strains which could swarm on lambda motility agar plates (14) and whose plasmids could be cut by HindIll, SmaI, and BglI. These plasmids were designated as pFD202 (derived from pFD2) and pFD301, pFD303, pFD306, pFD307, pFD311, pFD313, and pFD315 (all derived from pFD3). The ability of these plasmids to confer motility on C600 hsm hsr hag: :TnJO was estimated as the swarm zone size relative to that of the same strain carrying pBR322/hag93, which has the wild-type hag gene (17). Four of them (pFD301, pFD303, pFD306, and pFD307) conferred ca. 10% of wild-type swarming, three (pFD311, pFD313, and pFD315) conferred ca. 50%, and one (pFD202) conferred almost 100% (Fig. 1).HincIl restriction fragments of these plasmids were compared by agarose gel electrophoresis. Only the 1.7-kilobase fragment of pBR322/hag93, which includes the entire hag gene (17), was shortened in each of the deletion plasmids. From the length of these fragments, it was inferred that pFD202, pFD301, pFD303, pFD306, pFD307, pFD311, pFD313, and pFD315 were about 60, 540, 340, 320, 400, 250, 160, and 220 bases,...