IDENTIFICATION OF A SEGMENT OF THE ESCHERICHIA-COLI TSX PROTEIN THAT FUNCTIONS AS A BACTERIOPHAGE RECEPTOR AREA

The Escherichia coli outer membrane protein Tsx functions as a nucleos ide-specific channel and serves as the receptor for colicin K and a nu mber of T-even-type bacteriophages, including phage T6. To identify th ose segments of the Tsx protein that are important for its phage recep tor function, we devised a selection and screening procedure which all owed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were em ployed for the selection of phage-resistant derivatives of a strain ex pressing a tsx+-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-spec ific phage used for the initial mutant selection, cross-resistance aga inst a set of six different Tsx phages invariably occurred. With one e xception, these mutant Tsx proteins could still serve as a colicin K r eceptor. DNA sequence analysis of 10 mutant tsx genes revealed the pre sence of four distinct tsx alleles: two point mutations, an 18-bp dele tion, and a 27-bp tandem duplication. In three isolates, Asn-249 was r eplaced by a Lys residue (tsx-504), and in four others, residue Asn-25 4 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) r emoved six amino acids (residue 239 to residue 244) from the 272-resid ue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolat es) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx pro tein and the other mutant Tsx proteins, the Tsx-507 protein was cleave d by trypsin when intact cells were treated with this protease. The Ts x proteins encoded by the four tsx alleles still functioned in deoxyad enosine uptake in vivo, demonstrating that their nucleoside-specific c hannel activity was not affected by the alterations that caused the lo ss of their phage receptor function. The changes in the Tsx polypeptid e that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are d iscussed in terms of a model for the topological organization of the c arboxy-terminal end of the Tsx protein within the outer membrane.