Assembly of bacteriophage PRD1 spike complex: role of the multidomain protein P5.

The spike structure of bacteriophage PRD1 is comprised of proteins P2, P5, and P31. It resembles the corresponding receptor-binding structure of adenoviruses. We show that purified recombinant protein P5 is an elongated (30 x 2.7 nm; R(h) = 5.5 nm), multidomain trimer which can slowly associate into nonamers. Cleavage of the 340 amino acid long P5 with collagenase yields 2 fragments. The larger, 205 amino acid long C-terminal fragment appears to contain the residues responsible for the trimerization of the protein, whereas the smaller N-terminal part mediates the interaction of P5 with the pentameric vertex protein P31 (24 x 2.5 nm, R(h) = 4.2 nm). In addition, the presence of the N-terminal sequence is required for the formation of the P5 nonamer. The results presented here suggest that P5 and P31 form an elongated adaptor complex at the 5-fold vertexes of the virion which anchors the adsorption protein P2 (21 x 2.5 nm; R(h) = 4.1 nm). Our results also suggest that the P5 trimer forms a substantial part of the viral spike shaft that was previously thought to be composed exclusively of protein P2.

A new mutant class, made by targeted mutagenesis, of phage PRD1 reveals that protein P5 connects the receptor binding protein to the vertex.

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene V revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5(-) particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.

The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability.

IncP-type plasmids are broad-host-range conjugative plasmids. DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf). The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient. The Mpf complex acts as a receptor for IncP-specific phages such as PRD1. In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique. Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP. In addition it reduces the ability of the cells to accumulate K+. However, the Mpf complex does not dissipate the membrane voltage. The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.

Changes in host cell energetics in response to bacteriophage PRD1 DNA entry.

Double-stranded DNA bacteriophage PRD1 infects a variety of gram-negative bacteria harboring an IncP-type conjugative plasmid. The plasmid codes for the DNA transfer phage receptor complex in the cell envelope. Our goal was, by using a collection of mutant phage particles for which the variables are the DNA content and/or the presence of the receptor-binding protein, to obtain information on the energy requirements for DNA entry as well as on alterations in the cellular energetics taking place during the first stages of infection. We studied the fluxes of tetraphenylphosphonium (TPP+), phenyldicarbaundecaborane (PCB-), and K+ ions as well as ATP through the envelope of Salmonella typhimurium cells. The final level of the membrane voltage (delta psi) indicator TPP+ accumulated by the infected cells exceeds the initial level before the infection. Besides the effects on TPP+ accumulation, PRD1 induces the leakage of ATP and K+ from the cytosol. All these events were induced only by DNA-containing infectious particles and were cellular ATP and delta psi dependent. PRD1-caused changes in delta psi and in PCB- binding differ considerably from those observed in other bacteriophage infections studied. These results are in accordance with the presence of a specific channel engaged in phage PRD1 DNA transport.

Probing phage PRD1-specific proteins with monoclonal and polyclonal antibodies.

Four new specificity class MAbs against PRD1 proteins (P6, P7/14, P11, P18) and polyclonal antiserum against the minor capsid protein P5 were produced. The antibodies were used to analyze the phage protein distribution inside the host cell during infection as well as in the virion. The minor component of the capsid, P5, was shown to be located on the surface of the virion. The proteins responsible for particle infectivity were localized to the membrane fraction of the host cells. In addition, by detection with MAbs, genes encoding proteins P14 and P18 were positively localized on the PRD1 genome.