The Motion of a Single Molecule, the λ-Receptor, in the Bacterial Outer Membrane

Oddershede, Lene B.; Dreyer, Jakob Kisbye; Grego, Sonia; Brown, Stanley; Berg-Sørensen, Kirstine

doi:10.1016/s0006-3495(02)75318-6

Cited by 58 publications

(98 citation statements)

References 25 publications

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“…The earliest study of outer membrane protein mobility was performed by Oddershede et al [20], observing the diffusion of l-receptor (LamB), the maltodextrin transport channel in E. coli, using single-particle tracking in conjunction with a weak optical trap. The local short-time diffusion coefficient for the l-receptor was measured by attaching a 530 nm microsphere to the protein, and imaging the probe's motion at 25 Hz in the presence of weak laser optical tweezers.…”

Section: (A) Outer Bacterial Membranementioning

confidence: 99%

Single-molecule imaging in live bacteria cells

Ritchie

Lill

Sood

et al. 2013

Phil. Trans. R. Soc. B

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Bacteria, such as Escherichia coli and Caulobacter crescentus , are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

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Section: (A) Outer Bacterial Membranementioning

confidence: 99%

Single-molecule imaging in live bacteria cells

Ritchie

Lill

Sood

et al. 2013

Phil. Trans. R. Soc. B

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“…Although the term optical tweezers is widely accepted, the system is also often referred to as an optical trap. In some cases the optical tweezers trap beads attached to the biological system of interest, e.g., in the study of molecular motors (24) or bacterial outer membrane proteins (19). In other cases the object under investigation, e.g., a whole living cell, is directly trapped.…”

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confidence: 99%

Optical Tweezers Cause Physiological Damage to Escherichia coli and Listeria Bacteria

Rasmussen

Oddershede

Siegumfeldt

2008

Appl Environ Microbiol

158

112

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We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited morepronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.

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“…Single-molecule fluorescence experiments in live bacteria (35) have been limited by the small size of the cells (relative to the diffraction limited spatial resolution), as well as by the presence of cellular autofluorescence (primarily from various flavinoids) that can obscure the desired signal (36). Single proteins have also been tracked in bacteria by attaching them to polystyrene beads (37), but the large size of the beads (relative to the protein studied) makes them likely to perturb the motion of the protein.…”

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confidence: 99%

Visualization of the movement of single histidine kinase molecules in liveCaulobactercells

Deich

Judd

McAdams

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

129

149

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The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 ؎ 2 ؋ 10 ؊3 m 2 ͞s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.single molecule ͉ diffusion ͉ PleC ͉ enhanced yellow fluorescent protein T he inner membranes of bacterial cells contain proteins required for a wide variety of functions, including energy generation, solute transport, signaling, proteolysis, polar morphogenesis, chemotaxis, and cell division (1-3). The size of the diffusion coefficient (D) of these proteins in the membrane can affect their interactions with each other and with cytoplasmic proteins. For example, in Escherichia coli, the MinCDE system for locating the division plane is thought to require a difference in D between the membrane-associated and the cytoplasmic forms of the MinD and MinE proteins for its proper function (4-6). The D values of several cytoplasmic proteins have been measured in E. coli (7). Measurements of D for membrane proteins in eukaryotic cells, using fluorescence recovery after photobleaching (FRAP) (8), single gold bead tracking (9-11), and single-molecule tracking techniques (12, 13), have yielded values ranging from 5 ϫ 10 Ϫ3 to 500 ϫ 10 Ϫ3 m 2 ͞s. Each Caulobacter cell division produces a pair of distinct daughter cells (Fig. 1): a motile swarmer (SW) cell with a single flagellum located at a specific pole and a stalked (ST) cell possessing an adhesive holdfast at the end of the stalk, allowing it to attach to a surface (14, 15). The transmembrane histidine kinase PleC regulates polar organelle formation, motility, and asymmetric cell division in Caulobacter (16). PleC is a 90-kDa inner membrane protein, with four predicted transmembrane domains as obtained from TMPRED (www.ch.embnet.org͞ software͞TMPREDform.html). Cells with mutant PleC do not form stalks or pili and have paralyzed flagella (17)(18)(19). These mutant cells undergo symmetric cell division, producing two daughter cells of similar size, each possessing a paralyzed flagellum. By using conventional fluorescence microscopy, molecules of PleC were found to be localized to the flagellar pole of SW...

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The Motion of a Single Molecule, the λ-Receptor, in the Bacterial Outer Membrane

Cited by 58 publications

References 25 publications

Single-molecule imaging in live bacteria cells

Single-molecule imaging in live bacteria cells

Optical Tweezers Cause Physiological Damage to Escherichia coli and Listeria Bacteria

Visualization of the movement of single histidine kinase molecules in liveCaulobactercells

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