Microscopic 'clutch' puts flagellum in neutral

Caption: Scientists have learned a tiny molecular clutch disengages the flagellum's tail from the engine that powers its rotation. To see the full image, click the link below. Credit: Zina Deretsky, NSF Illustration courtesy of the National Science Foundation. Usage Restrictions: None.

BLOOMINGTON, Ind. -- A tiny but powerful engine that propels the bacterium Bacillus subtilis through liquids is disengaged from the corkscrew-like flagellum by a protein clutch, Indiana University Bloomington and Harvard University scientists have learned. Their report appears in this week's Science. Scientists have long known what drives the flagellum to spin, but what causes the flagellum to stop spinning -- temporarily or permanently -- was unknown.

"We think it's pretty cool that evolving bacteria and human engineers arrived at a similar solution to the same problem," said IU Bloomington biologist Daniel Kearns, who led the project. "How do you temporarily stop a motor once it gets going?"

The action of the protein they discovered, EpsE, is very similar to that of a car clutch. In cars, the clutch controls whether a car's engine is connected to the parts that spin its wheels. With the engine and gears disengaged from each other, the car may continue to move, but only because of its prior momentum; the wheels are no longer powered.

EpsE is thought to "sit down," as Kearns describes it, on the flagellum's rotor, a donut-shaped structure at the base of the flagellum. EpsE's interaction with a rotor protein called FliG causes a shape change in the rotor that disengages it from the flagellum's proton-powered engine.

The discovery of EpsE and its function was accidental. Kearns and colleagues were actually interested in learning more about the genes that cause individual cells of B. subtilis to cease wandering in solitude and take up residence in a massively communal, stationary assemblage called a biofilm. The stability of biofilms can be jeopardized by hyperactive bacterial cells whose flagella continue to spin.

"We were trying to get at how the bacterium's ability to move and biofilm formation are balanced," Kearns said. "We were looking for the genes that affected whether the cells are mobile or stationary. Although B. subtilis is harmless, biofilms are often associated with infections by pathogenic bacteria. Understanding biofilm formation may eventually prove useful in combating bacterial infections."

Once the scientists learned EpsE was involved in repressing flagellar motion, they devised two possible explanations for how EpsE acts. The first was that EpsE acts like a brake by pushing a non-moving part against a moving part and locking up the works. The other possibility, they imagined, was that EpsE acts like a clutch, disengaging one moving part from another. In this latter scenario, the engine can no longer drive flagellar spinning because key moving parts are no longer in contact. In this case, the flagellum would still have freedom of motion, listless as it might be.

To determine which hypothesis was correct, the scientists decided it best to let the tail wag the dog. They attached the tail end of the flagellum to a glass slide and examined the movement of the entire cell in the presence and absence of EpsE. In the absence of EpsE, the entire cell rotated once every five seconds. In the presence of EpsE, the cells stopped but could rotate passively, pushed by disturbances in the environment (Brownian motion). If EpsE acted like a brake, the cells would not have rotated at all.

The researchers also learned that when the cell begins producing EpsE, it takes about 15 minutes before the flagellar machinery is disabled.

"This makes a lot of sense as far as the cell is concerned," Kearns said. "The flagellum is a giant, very expensive structure. Often when a cell no longer needs something, it might destroy it and recycle the parts. But here, because the flagellum is so big and complex, doing that is not very cost effective. We think the clutch prevents the flagellum from rotating when constrained by the sticky matrix of the biofilm."

The discovery may give nanotechnologists ideas about how to regulate tiny engines of their own creation. The flagellum is one of nature's smallest and most powerful motors -- ones like those produced by B. subtilis can rotate more than 200 times per second, driven by 1,400 piconewton-nanometers of torque. That's quite a bit of (miniature) horsepower for a machine whose width stretches only a few dozen nanometers. ###

IU Bloomington Biology Research Associate Kris Blair is the paper's lead author. IUB undergraduate student Jared Winkelman and Harvard University microbiologists Linda Turner and Howard Berg also contributed to the report. It was funded with a grant from the National Science Foundation (Kearns) and the National Institutes of Health (Berg).

To speak with Kearns, please contact David Bricker, University Communications, at 812-856-9035 or brickerd@indiana.edu.

Contact: David Bricker brickerd@indiana.edu 812-856-9035 Indiana University

Tags: Nano or Nanotechnology and Nanotech

Trap and zap: Harnessing the power of light to pattern surfaces on the nanoscale

Caption: A technique developed by Princeton engineers allows the easy creation of nano-scale patterns on uneven surfaces and without the normal requirements of a vibration and oxygen-free environment. The black bar next to the Princeton shield is 2 microns long. Credit: Nature Nanotechnology/Princeton University, Usage Restrictions: Please credit Nature Nanotechnology.

Princeton engineers have invented an affordable technique that uses lasers and plastic beads to create the ultrasmall features that are needed for new generations of microchips. The method, which creates lines and dots that are 1,000 times narrower than a human hair, may enable the creation of biological computers as well as micromachines with applications in medicine, optical communications, computing and sensor technologies.

The technique, created by mechanical and aerospace engineering assistant professor Craig Arnold and graduate student Euan McLeod, is similar to poising a magnifying lens over a scrap of paper and angling the lens to focus sunlight and ignite the paper. In place of the lens, the researchers use a microscopic plastic bead floating in water to focus light from a powerful laser and burn designs onto a blank microchip. Their findings are reported online June 8 in the journal Nature Nanotechnology.

While others have passed laser light through various microscopic objects to pattern surfaces, they have struggled to maintain a consistent distance between the bead and the surface of the microchip. If this distance changes, the laser light is focused in different ways across the surface and the resulting pattern is inconsistent. Arnold and McLeod established an innovative way to ensure that the bead is always the same distance from the microchip, which allows them to draw on the surface with high levels of precision.

"One of the biggest challenges in probe-based nanopatterning is regulating the distance between your probe and the surface of the microchip," said Arnold. "We used a special laser to trap the bead and keep it close to the surface without touching it."

The researchers used the technique to "draw" features that were about 100 nanometers (a billionth of a centimeter) in size.

The key innovation is the use of a second, highly focused laser, which points directly down onto the bead. This intense light exerts a physical force on the bead, trapping it in the beam and pushing it down toward the surface. The surface pushes back with a constant force, and the bead settles at a height that balances the opposing forces. The original laser is then pulsed at the bead, which focuses the light to "zap" the surface directly below. By moving the bead along a computer controlled trajectory while repeating the laser pulse, a desired pattern is created.

The technique offers particular advantages on curved or irregular surfaces because the bead tracks the surface, moving up when there is a bump and dropping when it moves over a dip. While other fabrication techniques, such as electron-beam lithography, can also be used to pattern uneven surfaces, they are extremely expensive and must be performed in a vibration- and oxygen-free environment. The new Princeton technique can be performed in a regular environment, making it accessible for use with biological materials and other systems that require the presence of oxygen.

"The technique provides a very interesting new capability to expand laser-assisted nanofabrication without involving moving mechanical parts and related hardware complications," said Costas Grigoropoulos, mechanical engineering professor at University of California-Berkeley. "I do expect that this novel technique will advance nanopatterning since it offers an elegant and highly effective means for parallel, optically driven and controlled nanofabrication."

In addition to burning away parts of a chip, Arnold and McLeod's method has the potential to deposit materials on surfaces, rather like gold-plating. This could provide a new means of creating three-dimensional structures, including miniscule guides that manipulate light and nanoscale electrical-mechanical devices. Such devices have many potential uses in ultrasmall sensor systems and low-power computer processors.

"In the future, we imagine the use of multiple beads of different shapes and sizes -- in essence a nanopatterning toolkit -- for researchers to pick and choose during the course of fabrication," said Arnold. He and McLeod are currently working to pattern a surface using an array of many beads moving in parallel, each trapped and controlled by a different laser beam. ###

The research was supported by Princeton University and the Air Force Office of Scientific Research.

Contact: Steven Schultz sschultz@princeton.edu 609-258-3617 Princeton University, Engineering School

Tags: Nano or Nanotechnology and Nanotech