Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association.

Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

Conservation of the N-terminus of some phage tail proteins.

To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.

Insertional mutagenesis in the tailspike protein of bacteriophage P22.

The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for recognition and hydrolysis of Salmonella typhimurium host receptors. Once properly folded, TSP shows an unusual stability to temperature and detergent denaturation, prompting the analysis of TSP as a framework for the positioning of heterologous protein segments. We have explored the flexibility of inner sites and both amino and carboxy termini to accommodate foreign peptides for phage display. In the examined inner sites, TSP is extremely sensitive to minor sequence modifications, the folding intermediates being rapidly degraded. However, both the amino and carboxy termini are tolerant to peptide fusions, rendering stable and functional chimeric proteins. Surprisingly, the amino terminus, which connects the tail to the neck structure, can accept large peptide fusions, and the foreign amino acid stretches are solvent-exposed and highly antigenic on assembled, infectious virus particles.

Sequence comparison of the genes for immunity, DNA replication, and cell lysis of the P22-related Salmonella phages ES18 and L.

Complementation and hybridization experiments with the generalized transducing Salmonella phages P22, ES18 and L revealed strong similarity between the phages L and P22; the genome of ES18 shows a mosaic structure. About half of its genome, including the early genes, is similar or completely homologous to P22; the other half of the morphologically different ES18 does not show any similarity to P22 nor to E. coli phage lambda. Sequence comparison of the early genes has confirmed that the C-immunity region of ES18 is identical with that of P22, whereas the same region of phage L shows poor (repressor gene) or no similarity. The 5'-terminus of the DNA replication gene 12 of ES18, however, is homologous to the same section of gene O of phage lambda. The lysis genes of ES18 again are identical to those of P22; only gene 15 is mosaic-like and has more similarity to gene Rz of phage lambda. These results will be discussed in terms of the theory of modular genome organization.

Frequency of generalized transducing phages in natural isolates of the Salmonella typhimurium complex.

From 85 natural isolates of the Salmonella typhimurium complex, including the Salmonella reference collection A (P. Beltran, S. A. Plock, N. H. Smith, T. S. Whittam, D. C. Old, and R. K. Selander, J. Gen. Microbiol. 137:601-606, 1991), 65 strains (76.5%) released 71 different temperate phages. Forty-three (93.5%) of 46 tested phages were able to transduce the chromosomal markers his+ and trp+ and the cloning vector pBR325.