The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.

The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression.Filamentous bacteriophage is a nonlytic DNA virus that infects Escherichia coli cells harboring an F episome (19,20,26). In order for bacteriophage infection to occur, including the translocation of the single-stranded phage DNA over the cell membrane, the expression of two different receptor systems in the gram-negative host cell is critical. The primary receptor, i.e., the F pilus, mediates the adsorption of the phage to the bacterial cell (6,12). This interaction can take place at a long distance from the cell surface, since the pilus molecule extrudes away from the bacteria. Through the depolymerization of the pilus, the bacteriophage is brought close to the cell surface of the host, where the interaction with the coreceptor takes place (5, 31). The coreceptor, i.e., the TolQRA complex, is localized in the inner membrane and the periplasmic space and is attached to the inside of the outer membrane (9, 16). It was recently shown that it is the C-terminal part of the TolA domain that interacts with the bacteriophage adsorption protein (7,27). Upon binding to the TolA domain, the viral DNA translocates into the host cell by an as-yet-unknown mechanism.Expression of the gene 3 viral adsorption protein, g3p, on the surface of the bacteriophage is essential for normal infection to occur (1). The minor coat protein g3p is located together with g6p in three to five copies at one end of the filamentous phage (10). Mature g3p consists of 406 amino acid residues separated in several distinct regions (2). The N-terminal part can be divided into two different domains, N1 and N2, mediating penetration and adsorption during infection, respectively (1, 30). The C-terminal part is responsible for the interaction with g6p, thereby anchoring the g3p in the membrane of the phage coat (1, 14, 21). Furthermore, the g3p has a crucial role in the assembly of the filamentous phage, since in the absence of g3p, the proper termination of the assembly and the following release of the phage will not take place (24).Two glycine-rich linker regions divid...

Selective infection of phage is when the bacterial infection depends on the specific molecular interaction between an antigen and a phage-displayed protein sequence such as an antibody. Engineering of the normal infection into pathways, directed by a specific protein--protein interaction, has raised several mechanistic questions. Here, we address the type of display and the affinity between the interacting pairs. The deleted phage R408d3 was used for the first time in selective infection and was shown to exhibit a superior performance compared to the VCSM13 phage. Furthermore, the affinity between the interacting pairs also affected the selective infection process and a correlation between affinity and infection efficiency was detected, thus implying that selective infection is the method of choice for selection of rare high-affinity interactions in molecular libraries.

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