![]() ![]() Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Thus, transcription in the presence of antibiotic induces vmlR expression. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. Here authors describe high resolution cryo-EM structures of CcmABCD in multiple states and propose a hypothetic model of heme trafficking, heme transfer and release of holoCcmE. Bacterial ABC transporter complex CcmABCD, a part of cytochrome c maturation system, transfers heme from one binding partner (CcmC) to another (CcmE). CcmABCD represents an ABC transporter complex using the energy of ATP hydrolysis for the transfer of heme from one binding partner (CcmC) to another (CcmE). Based on our structures combined with functional studies, we propose a hypothetic model of heme trafficking, heme transfer to CcmE, and ATP-dependent release of holoCcmE from CcmABCD. We locate the ATP-binding site in CcmA and the heme-binding site in CcmC. We describe high resolution cryo-EM structures of CcmABCD in an unbound form, in complex with inhibitor AMP-PNP, and in complex with ATP and heme. A large membrane complex, CcmABCD has been proposed to carry out this transport and linkage to CcmE, yet the structural basis and mechanisms underlying the process are unknown. Since all c-type cytochromes are periplasmic, heme is first transported to a periplasmic heme chaperone, CcmE. The cytochrome c maturation system I, consisting of eight membrane proteins (CcmABCDEFGH), results in the attachment of heme to cysteine residues of cytochrome c proteins. Cytochromes c use heme as a cofactor to carry electrons in respiration and photosynthesis. ![]()
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