RESUMO
The DctSR two-component system of Bacillus subtilis controls the expression of the aerobic C4-dicarboxylate transporter DctA. Deletion of DctA leads to an increased dctA expression. The inactivation of DctB, an extracellular binding protein, is known to inhibit the expression of dctA. Here, interaction between the sensor kinase DctS and the transporter DctA as well as the binding protein DctB was demonstrated in vivo using streptavidin (Strep) or His protein interaction experiments (mSPINE or mHPINE), and the data suggest that DctA and DctB act as cosensors for DctS. The interaction between DctS and DctB was also confirmed by the bacterial two-hybrid system (BACTH). In contrast, no indication was obtained for a direct interaction between the transporter DctA and the binding protein DctB. Activity levels of uptake of [(14)C]succinate by bacteria that expressed DctA from a plasmid were similar in the absence and the presence of DctB, demonstrating that the binding protein DctB is not required for transport. Thus, DctB is involved not in transport but in cosensing with DctS, highlighting DctB as the first example of a TRAP-type binding protein that acts as a cosensor. The simultaneous presence of DctS/DctB and DctS/DctA sensor pairs and the lack of direct interaction between the cosensors DctA and DctB indicate the formation of a tripartite complex via DctS. It is suggested that the DctS/DctA/DctB complex forms the functional unit for C4-dicarboxylate sensing in B. subtilis.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Ácidos Dicarboxílicos/metabolismo , Proteínas de Membrana/metabolismo , Bacillus subtilis/classificação , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ácidos Dicarboxílicos/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/genética , Plasmídeos , Ligação ProteicaRESUMO
Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Periplasma/metabolismo , Proteínas Quinases/genética , Transdução de Sinais , Sistemas de Secreção Bacterianos/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Proteínas de Membrana/metabolismo , Pressão Osmótica , Ligação Proteica , Proteínas Quinases/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismoRESUMO
Membrane proteins are essential for cell viability and are therefore important therapeutic targets(1-3). Since they function in complexes(4), methods to identify and characterize their interactions are necessary(5). To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE(6). This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane- SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.