Transport mechanisms of bacterial membrane proteins analyzed by advanced fluorescence microscopy

Abstract

Transporter proteins are integral membrane proteins and enable the translocation of hydro- or amphiphilic substrates over cell membranes. This study focuses on two such systems: The lipid II flippase and the tripartite adensosine triphosphate (ATP)-independent periplasmic (TRAP) transporters. <br/> The lipid II flippase is essential for cell wall synthesis in bacteria, as this protein mediates the translocation of the cell wall precursor lipid II from cyto- to periplasm. However, the exact identity of this protein remains a topic of discussion. <br/> TRAP transporters scavenge and import substrates from host cells and, thereby, contribute to the virulence of different pathogens. These transporters consist of a substrate binding protein (SBP), also called P-domain, and two transmembrane domains (Q- and M-domain). The SBP diffuses freely in the bacterial periplasm and delivers the substrate to the transmembrane domains. <br/> The complexity of biological membranes makes it difficult to study single proteins, however, membrane proteins need to be in hydrophobic environments to be active. Therefore, advanced fluorescence microscopy was used to investigate both processes in artificial membranes. To this end, a modified formation pathway for giant unilamellar vesicles (GUVs) was developed, which enabled a high GUV yield in physiological salt concentration solutions while allowing a simultaneous reconstitution of membrane proteins. Subsequently, GUVs and large unilamellar vesicles (LUVs) were characterized as model systems. <br/> The putative lipid II flippases MurJ, RodA and FtsW from <em>Staphylococcus aureus</em> (Sa) were tested for their lipid II flipping activity in presence and absence of a membrane potential. MurJ was found to exhibit lipid II flipping, in contrast to the other tested proteins. MurJ's activity was increased in presence of a membrane potential. <br/> Sialic acid import by SiaPQM from <em>Haemophilus influenzae</em> (Hi) was visualized in alternating-laser excitation (ALEX) single-molecule (sm) Förster resonance energy transfer (FRET) measurements as well as in ensemble FRET measurements. Both import assays revealed that the SBP is strictly required for successful import. Furthemore, a sodium gradient proved to be essential as a driving force for the import. If no sodium was present, binding between HiSiaP and HiSiaQM was attenuated. Finally, important regions in HiSiaPQM were identified that were necessary for substrate import. One of these regions was a periplasmic loop of HiSiaQM which could potentially be utilized to trigger a "push-to-release"-mechanism in HiSiaP, mediating successful substrate transfer.