The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases

The temperate bacteriophage Sf6 infects Shigella flexneri strains of seroty pe X or Y, converting them into serotypes 3a or 3b, respectively. The tails pike protein (TSP) of Sf6 possesses endo-1,3-alpha-L-rhamnosidase (endorham nosidase) activity which results in cleavage of the lipopolysaccharide O-an tigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene g (encoding P22 TSP) DNA probe hybridized with restriction fragment Pstl-7 of Sf6. DNA sequencing and anal ysis of Pstl-7 and the adjacent Pstl-8 fragment revealed an open reading fr ame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homolog y to the bacteriophage P22 TSP N-terminal head-binding domain. High conserv ation of key residues was suggestive of similar secondary and tertiary N-te rminal protein structure and a similar function of the Sf6 TSP in this regi on. In addition, an amino acid sequence motif (DFGX(3)DGX(6)AX(3)A) was ide ntified between residues 164 and 184 which was also found to exist in vario us prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacte riophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by L-[S-35]methionine labelling and SDS-PAGE), Assay of h eat-treated cytoplasmic extracts containing the ORF1-encoded protein by inc ubation with whole Sh, flexneri Y cells demonstrated that O-antigen hydroly sis activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific and preferential activity for long-chain Sh, flexneri serotype X or Y O-antigen,, cleavage of which resulted in the release of oligosacchari de fragments, consistent with octasaccharides in size, as detected by fluor ophore-assisted carbohydrate electrophoresis (FACE).