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

• Published in: Microbiology (Society for General Microbiology) 1999-07, Vol.145 (7), p.1649-1659
• Main Authors: Chua, James E. H, Manning, Paul A, Morona, Renato
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteriophages - enzymology; Base Sequence; Biological and medical sciences; Blotting, Southern; Carbohydrate Sequence; Cloning, Molecular; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA, Viral - analysis; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Microbiology; Molecular Sequence Data; Phage P22; Phage Sf6; Polymerase Chain Reaction; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Restriction Mapping; Sequence Analysis, DNA; Shigella flexneri - virology; Viral Tail Proteins - chemistry; Viral Tail Proteins - genetics; Viral Tail Proteins - metabolism; Virology
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-145-7-1649

Department of Microbiology and Immunology, University of Adelaide, Adelaide, South Australia, Australia 5005 Author for correspondence: Renato Morona. Tel: -61 8 8303 4151. Fax: -61 8 8303 4362. e-mail: [e-mail] ABSTRACT The temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3- - L -rhamnosidase (endorhamnosidase) activity which results in cleavage of the lipopolysaccharide O-antigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene 9 (encoding P22 TSP) DNA probe hybridized with restriction fragment Pst 1 -7 of Sf6. DNA sequencing and analysis of Pst l-7 and the adjacent Psr l-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP N-terminal head-binding domain. High conservation of key residues was suggestive of similar secondary and tertiary N-terminal protein structure and a similar function of the Sf6 TSP in this region. In addition, an amino acid sequence motif (DFGX 3 DGX 6 AX 3 A) was identified between residues 164 and 184 which was also found to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by L -[ 35 S]methionine labelling and SDS-PAGE). Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole Sh. flexneri Y cells demonstrated that O-antigen hydrolysis 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 oligosaccharide fragments, consistent with octasaccharides in size, as detected by fluorophore-assisted carbohydrate electrophoresis (FACE). Keywords: tailspike protein. cloning, motif, O-antigen, endorhamnosidase activity The GenBank accession number for the sequence determined in this work is AF128887.