Lytic conversion of Escherichia coli by bacteriophage T5: blocking of the FhuA receptor protein by a lipoprotein expressed early during infection.

The nucleotide sequence of the region between the oad gene, encoding the host specificity protein, and the right-terminal repetition of bacteriophage T5 DNA was determined. Five small open reading frames, the first of which was called llp, were detected, which apparently formed an operon transcribed from a promoter that overlapped the oad promoter. Both promoters were confirmed by primer extension assays. Using mRNA isolated at different times after T5 infection, the llp and oad promoters were identified as early and late promoters, respectively. The N-terminus of the llp gene product possess a signal sequence and a processing site characteristic of lipoproteins. After subcloning and expression of llp, its product Llp was identified as a 7.8 kDa polypeptide. Acylation of Llp was confirmed by addition of globomycin, which resulted in the accumulation of the unprocessed precursor form. FhuA+ cells synthesizing Llp were resistant to phage T5. Resistance was caused by inhibition of adsorption of T5 to its FhuA receptor protein. Resistance could be overcome by derepression of fhuA transcription, suggesting a blocking of FhuA by direct interaction with Llp. Since Llp-mediated T5 resistance has several aspects in common with the phenomenon of lysogenic conversion, we suggest that it should be called lytic conversion.

Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein.

Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene. We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7 promoter. This segment contains an open reading frame that can encode a protein of the appropriate size. Its deduced amino acid sequence does not closely resemble that of any other protein in the database. The sequence upstream of the open reading frame shows typical characteristics of a promoter region with two overlapping, divergently orientated promoters.

Location of genes D16, D17, and N4 encoding tail proteins on the physical map of bacteriophage T5.

Bacteriophage T5 DNA fragments were cloned into plasmid pBR322. Recombinant plasmids complementing T5 amber mutants were isolated, and used as hybridization probes with T5 DNA in Southern blots. Employing this approach the three T5 genes D16, D17, and N4 were mapped with respect to the physical map of T5, and shown to be located at 74%, 72%, and 82% of the genome, respectively.

Cloning and expression of the ltf gene of bacteriophage T5.

The unique 5-kilobase BamHI fragment of bacteriophage T5 was cloned into plasmid pBR322. Location of the intact ltf gene on the cloned fragment was demonstrated by complementation of the ltf mutation of phage T5hd-2, identification of a plasmid-coded polypeptide of the same molecular weight as the polypeptide forming the L-shaped tail fibers, which binds to anti-T5 antibodies; and analyses of transposon Tn1000 insertions.