RESUMO
The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Modelos Moleculares , Fosfatidiletanolaminas/química , Proteínas Quinases/química , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Acholeplasma laidlawii/enzimologia , Acholeplasma laidlawii/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cardiolipinas/química , Cardiolipinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Propriedades de SuperfícieRESUMO
Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study, we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present blue native-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in the knowledge of this organism's biology. On a more limited scale, we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecalis, which is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.
Assuntos
Proteínas de Bactérias/análise , Membrana Celular/química , Enterococcus faecalis/química , Proteoma/análise , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
