Browsing by Subject "DNA translocation"

Now showing 1 - 1 of 1

Protein interactions between the bacteriophage T4 tail tip and the inner membrane of Escherichia coli
(2025) Wenzel, Sabrina; Kuhn, Andreas
The infection process of bacteriophage T4 represents a fascinating series of orchestrated events, involving highly coordinated conformational changes and molecular interactions. While the initial stages, such as adsorption, host receptor recognition, and tail sheath contraction, are well-characterized (Islam et al., 2019; Leiman et al., 2004), the molecular mechanism underlying the translocation of the T4 genome across the inner host membrane remains poorly understood. The transport of the T4 DNA across bacterial membranes is particularly challenging due to its hydrophilic, polyanionic nature and the need to traverse the hydrophobic barriers of bacterial membranes. This step is critical for infection success, as it must occur without disrupting the host cell’s membrane potential or metabolic energy supply, which are essential for phage propagation. The central spike complex, comprising gp5Cβ and gp5.4, is the first component to enter the periplasm upon contraction of the T4 phage tail. This study aimed to investigate the interactions between the T4 phage tail tip proteins gp27, gp5, and gp5.4 and periplasm and inner membrane components of Escherichia coli. The first part of this study examined the behavior of the T4 central spike complex, specifically the gp5Cβ-gp5.4 component, after penetrating the outer membrane of E. coli. The spike complex, composed of three gp5Cβ and one gp5.4 subunit, punctures the outer membrane during tail contraction and enters the periplasm. To explore potential interactions with periplasmic components, in vitro binding assays using purified gp5Cβ-gp5.4 complexes and E. coli spheroplasts were performed. Results indicated that the spike complex lacked significant affinity for membrane bilayers, consistent with its structural properties, which lack hydrophobic or amphipathic regions. Cross-linking experiments with spheroplasts revealed a transient interaction between the spike complex and the periplasmic chaperone PpiD. This interaction was confirmed using proteoliposomes reconstituted with purified PpiD, which showed enhanced binding of the spike complex compared to control liposomes. PpiD is a periplasmic chaperone anchored to the inner membrane and is known for its role in stabilizing unfolded proteins. It is hypothesized that PpiD transiently interacts with the spike complex, stabilizing it or facilitating its positioning near the inner membrane for subsequent stages of infection. The biological relevance of PpiD was assessed using an efficiency of plating (EOP) assay, which revealed a reduction in infection efficiency in PpiD deletion mutants, with plaque formation decreasing to approximately 80% of wild type levels. This suggests that while PpiD is not essential for infection, it provides a supportive role, potentially complemented by other periplasmic chaperones. The second part of the study focused on the tail tip protein gp27 and its interactions with the inner membrane. Gp27, which forms a trimeric structure associated with gp5, is proposed to play a crucial role in forming a channel for DNA translocation. To investigate its potential binding to inner membrane proteins, T4 phage particles carrying His-tagged gp27 were constructed, and cross-linking experiments were performed in vivo. These studies identified several host proteins that interact with gp27, including DamX and PpiD. DamX, a cell division protein involved in peptidoglycan remodeling and septal ring stabilization, was identified as a key binding partner of gp27. Cross-linking and affinity purification confirmed this interaction, and subsequent infection assays demonstrated a significant reduction in T4 infection efficiency to 60% in DamX deletion mutants. These findings underscore the critical role of DamX in facilitating phage DNA translocation across the inner membrane. The study suggests that DamX may provide a structural or functional scaffold, stabilizing the phage-host interface during infection. PpiD, which was also identified in the first part of the study, was co-purified with gp27, highlighting its involvement in multiple stages of the infection process. Its role in stabilizing the phage at the inner membrane or facilitating structural rearrangements required for DNA translocation warrants further investigation. In addition to identifying these interactions, the study explored the functional implications of DamX and PpiD during infection. Complementary experiments demonstrated that both proteins are not only important for initial binding but may also be involved in facilitating the structural transitions required for successful DNA translocation.