Abstract
Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-angle X-ray scattering data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.
Highlights
The three-layered structure of the Gram-negative cell envelope, comprising the outer membrane, inner membrane, and intervening peptidoglycan, is a formidable defensive barrier
These observations are consistent with colicin E3 (ColE3) making additional interactions with immunity protein 3 (Im3) outside of the ribosomal ribonuclease (rRNase) domain, which are predicted to be absent from Colicin E9 (ColE9)
Our structure for the ColE9– immunity protein 9 (Im9) complex, only the second intact colicin nuclease structure after the ColE3–Im3 complex [21], shows how the conserved scaffold of T- and R-domains presents the E9 DNase–Im9 complex in a different orientation to that of the E3 rRNase–Im3 complex where the Im protein is sandwiched between the T- and C-domains
Summary
The three-layered structure of the Gram-negative cell envelope, comprising the outer membrane, inner membrane, and intervening peptidoglycan, is a formidable defensive barrier. Antimicrobial peptides and proteins from competing bacteria can breach these defences. Such competition systems are an integral part of the cohabitation of bacteria in mammalian hosts and are often associated with pathogenesis [1]. Three forms of protein-mediated bacterial antagonism are known to exist: bacteriocins ( protein antibiotics), which are released into the extracellular environment, contactdependent inhibition (CDI), and type VI secretion [2,3,4]. All three forms of antagonism result in death of a targeted cell by a toxin to which the producing strain is usually resistant. Many similarities are beginning to emerge between the modes of action of bacteriocins, CDIs, and the type VI secretion system, most notably in the types of cytotoxic activities delivered to the susceptible cell
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