1887

Abstract

The temperate bacteriophage Sf6 infects strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3-α--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 (encoding P22 TSP) DNA probe hybridized with restriction fragment 1 of Sf6. DNA sequencing and analysis of l-7 and the adjacent 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 (DFGXDGXAXA) 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 -[S]methionine labelling and SDS-PAGE). Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole 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 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).

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-145-7-1649
1999-07-01
2019-10-15
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-145-7-1649
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error