RESUMO
Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.
Assuntos
Bacteriocinas/química , Colicinas/química , Escherichia coli/metabolismo , Porinas/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Sítios de Ligação , Colicinas/genética , Colicinas/metabolismo , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Porinas/genética , Porinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , TermodinâmicaRESUMO
Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σ(R) preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA-σ(R) complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all σ(R)-binding residues are sequestered back into its hydrophobic core, releasing σ(R) to activate transcription of anti-oxidant genes.
