The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

Abstract

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...

The Mechanism of Bacterial Infection by Filamentous Phages Involves Molecular Interactions between TolA and Phage Protein 3 Domains

“…2). The inhibitory effect of proteins N1, N2 and N1L1N2 found in this study correlates with previous reports (27,33). The domains of the TolA protein, however, were not able to inhibit normal phage infection (data not shown).…”

“…An interaction with TolAII in the periplasm after the pilus has depolymerized would help g3p orient itself in the inner membrane, in a way that would facilitate its reuse and its possible role in the formation of a pore. However, it seems that the N2-TolAII interaction is not necessary for infection, as demonstrated by our previous finding that phage lacking N2 is capable of infecting pilus-bearing bacteria at almost the same frequency as wt phage (33). Also, the presence of the TolAII domain is not an absolute requirement for phage infection but contributes to the efficiency of TolA function in infection (9).…”

“…In a recent study we showed that the first glycine-rich linker region of g3p (L1) had an active role in F-pilus-dependent infection (33). In another study, the CT of g3p was found to interact specifically with the two N-terminal domains (N1L1N2), leading to a stable compact conformation of g3p on the phage, which is expected to unravel during infection (8).…”

“…The construction of N1L1N2 has been described (32). For the construction of N1 and N2, see the work of Nilsson et al (33).…”

Selective infection of E. coli as a function of a specific molecular interaction

“…pJARA131; for details on genetic markers see Nilsson et al, 2000). The cultures were maintained in Terrific Broth (TB) medium (10 g Bacto tryptone, 1 g yeast extract, 4 g NaCl and 1 g glucose per liter) supplemented with appropriate antibiotic and 1% of glucose.…”