Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.bioenergetics | membrane transport | FepA | iron transport F rom its importance in aerobic metabolism, iron is essential to most pro-and eukaryotes and therefore is a determinant of bacterial disease. Its sequestration by transferrin, lactoferrin, ferritin, heme compounds, and lipocalins defends animal cells, fluids, and tissues by "nutritional immunity" (1). However, efficient pathogens overcome this barrier and capture Fe 3+ either by producing siderophores (2) or by directly removing the metal from eukaryotic proteins (3). The trilaminar cell envelope of Gram-negative bacteria, composed of inner membrane (IM), outer membrane (OM), and the periplasm between them, contains protein components that confer the uptake of metabolic solutes, including sugars, amino acids, nucleotides, vitamins, and metals such as iron (4, 5). Enigmatic OM active transporters acquire metal complexes (ferric siderophores, heme, vitamin B 12 ) from the environment (6). The OM protein ferric enterobactin permease A (FepA), for example, internalizes the siderophore ferric enterobactin (FeEnt) (7). It is typical of many homologous metal transporters in commensal and pathogenic organisms. These uptake reactions also require TonB (8), a cell envelope protein that long ago was proposed to transduce energy (9-12). However, many questions exist about TonB's mediation of iron uptake, including its physical mechanism and its relationship to bioenergetics. Proton motive force (PMF) may drive OM active transport (6-8), but the mode of energy transmission to the OM and TonB's potential role in it are unknown. We addressed these topics by characterizing the localization of TonB in the cell membranes, by monitoring its physical motion, and by determining the dependence of its movements on metabolic energy and the additional IM proteins ExbBD.Iron chelates bind to their OM transporter...

Organic dyes usually exhibit enhanced photostability when trapped inside sol-gel silicates. The enhanced photostability is attributed to the reduction of intramolecular motions that facilitate photodegradation. We report the simultaneous detection of mobility and photostability of sol-gel encapsulated didodecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) using single molecule spectroscopy. Fluorescence from DiI was resolved into parallel and perpendicular polarization components and separately detected. On the basis of the calculated fluorescence polarization, single DiI molecules were classified into "tumbling" and "fixed". Out of 212 molecules investigated, 52% were found to be fixed. For the first time, the mobility of a guest molecule in sol-gel silicate can be directly correlated with its own photostability. Both tumbling and fixed molecules have shown to exhibit nonuniform photostability, indicative of the very heterogeneous guest-host interactions within each subgroup. The survival lifetimes for the majority of the tumbling and fixed molecules were found to be 4.3 and 13.1 s, respectively, demonstrating unequivocally that fixed molecules exhibit a higher photostability than tumbling molecules. These results are in accordance with a recent study on rhodamine B encapsulated in dried sol-gel silicates.

We investigate the effect of Coulombic interactions on the mobility of rhodamine 6G (R6G) and Oregon Green 514 (ORG) in sol−gel silicates by measuring their mobility distributions using single molecule polarization measurements. While R6G is a cationic dye, ORG (pK a = 3.69 ± 0.08) is an anionic dye at neutral pH. The presence of more tumbling ORG molecules in sol−gel silicates indicates that R6G and ORG experience opposite Coulombic interactions with a predominately anionic silica sol−gel surface. On the other hand, the fact that tumbling ORG only represents a minor portion of ORG investigated, even at pH 7, clearly illustrates that Coulombic interaction alone does not control the mobility of an encapsulated guest molecule in sol−gel silicates.

Easy to use and easy to produce biosensors would have a huge range of applications. To reach this goal many see the incorporation of a protein into a sol-gel network as one of the most viable options. The current most prevalent technique of predoping presents inherent limits on the concentration possible for the resulting thin film. In this study we demonstrate a new process utilizing the newly developed kinetic doping method to load silica sol-gel thin films with cytochrome C (CytC) and horseradish peroxidase (HRP). Both enzymes are shown to successfully load and have a concentration increase over their original loading solution by factors of 1300× and 2600×, respectively. Furthermore, each enzyme once loaded retained the ability to act as a catalyst for the detection of hydrogen peroxide. Ultimately the CytC- and HRP-loaded thin films were found to have enzyme concentrations of 11 ± 1 mM and 6.0 ± 0.4 mM, respectively, a considerable step up from any doping method reported in the past.