RESUMO
Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.
Assuntos
Sistemas de Secreção Tipo III/química , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades Proteicas , Salmonella typhimurium/química , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/ultraestruturaRESUMO
In the version of this article initially published, the PDB code associated with the study was given as 6F2E but should have been 6F2D in Table 1 and the data availability statement. The error has been corrected in the HTML and PDF versions of the article.
RESUMO
Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.
Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Complexos Multiproteicos , Eletroforese em Gel de Poliacrilamida Nativa , Proteínas de Bactérias/química , Western Blotting , Fracionamento Celular , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Imunoprecipitação , Proteínas de Membrana/química , Complexos Multiproteicos/química , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Salmonella typhimuriumRESUMO
Salmonella spp. are accountable for a large fraction of the global infectious disease burden, with most of their infections being food- or water-borne. The phenotypic features and adaptive potential of Salmonella spp. appear to be driven to a large extent by mobile or laterally acquired genetic elements. A better understanding of the conduct and diversification of these important pathogens consequently requires a more profound insight into the different mechanisms by which these pivotal elements establish themselves in the cell and affect its behavior. This review, therefore, provides an overview of the physiological impact and domestication of the Salmonella mobilome.
Assuntos
Adaptação Biológica , Evolução Molecular , Transferência Genética Horizontal , Salmonella/genética , Salmonella/patogenicidade , Salmonella/fisiologia , VirulênciaRESUMO
Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage-host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium-phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.
RESUMO
The Mrr protein of Escherichia coli K12 is a cryptic Type IV restriction endonuclease whose activity appears to be triggered by high pressure stress. In this report we used high pressure to isolate and analyze several Mrr mutants, and generated a new structural model of the Mrr protein. The activity of a number of spontaneous and strategically constructed Mrr mutants is discussed in the light of this model, providing a first insight into the structure-function relationships of the Mrr enzyme.
