Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

Abstract

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host’s existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA. IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

The multifarious roles of Tol-Pal in Gram-negative bacteria

“…Phages appear not to require Tol-Pal be coupled to the proton motive force for cell entry (Samire et al . 2020 ). Involvement of the PMF in colicin translocation remains controversial (Cramer, Sharma and Zakharov 2018 ).…”

First Report of Filamentous Phages Isolated from Tunisian Orchards to Control Erwinia amylovora

“…Typically, filamentous phages enter new hosts via pili on the cell surface [ 15 ]. The internalization of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system [ 16 ].…”

The Tol–Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate

“…Deletions were confirmed by PCR experiments according to the recommended strategy [25]. Plasmids ptolQR, ptolQ T145A R and ptolQR D23C [18] were used for complementation experiments.…”