Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes

Abstract

Each of the two mutants isolated from a fliC (=hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one more mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp.(and used for insertion of epitope-specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by λ gt11 with the N-terminal part of gene lacZ joined to a restriction fragment coding for residues 145–391 of flagellin gave the same patter of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105bp deletion as the first-isolated mutant; the fifth had a 72bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar antigen alleles.