The proton‐motive force is required for translocation of CDI toxins across the inner membrane of target bacteria

Abstract

Summary Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI+ inhibitor cells, binds to receptors on neighboring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm.

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

“…S2) (16). Therefore, we asked whether CdiA-CT EC869 tRNase activity is detectable in the tsf mutants.…”

“…Total RNA was extracted with guanidinium isothiocyanate (GITC)-phenol as described (40,41) and resuspended in 10 mM sodium acetate (pH 5.2), which stabilizes the aminoacyl linkage (42). Total RNA (5 μg) was analyzed by Northern blot hybridization using [ 32 P]-labeled oligonucleotides 3894, CH452, CH800, CH801, and CH1417 as probes (16). S30 extracts were prepared from E. coli CH2016, DL8530, and DL8546 cells grown to OD 600 ∼ 0.5.…”

“…CdiA-CT II Bp1026b preferentially cleaves within the aminoacyl acceptor stem of tRNA Ala to block translation (15). Other unrelated CdiA-CT toxins from E. coli isolates EC869 and 3006 also cleave tRNA acceptor stems but are specific for tRNA Gln and tRNA Ile , respectively (5,16). Thus, interbacterial competition has exerted a selective pressure to evolve diverse tRNase toxins with distinct specificities.…”

Interbacterial signaling via Burkholderia contact-dependent growth inhibition system proteins

“…BcpAIOB-mediated signaling could occur in recipient bacteria via several potential mechanisms. Toxin delivery into recipient cells requires translocation across both the outer and inner membranes (18), and one possibility is that the gene expression changes observed represent responses to membrane damage sustained during toxin entry. Indeed, envelope stress is known to trigger biofilm formation in other organisms (33,34).…”

“…Most BcpA-CT and CdiA-CT polypeptides function as nucleases in vitro and are sufficient to mediate cell death when produced intracellularly (12)(13)(14). According to the current model, BcpA-CT or CdiA-CT toxins are delivered to recipient bacteria upon cell-cell contact, exploiting outer membrane and inner membrane proteins on the recipient cell for entry to the cytoplasm (17,18). CDI + bacteria are protected from autoinhibition by production of an immunity protein, BcpI or CdiI.…”