Lab on a Chip Technology to Investigate how Bacteria Move

Awaking in the middle of the night, every tiny sound—a creaking floorboard, the drip of a tap, the quiet breathing of the murderer hiding in the wardrobe—can appear magnified. Yet, during the day, when background noise is higher, we don’t notice these same sounds. It’s not that they aren’t there, or even that other sounds are drowning them out, but rather that our sense of hearing calibrates itself to the background levels of noise. In the dead-silence of the night, our ears are far more sensitive to a tiny increase in sound which we would normally ignore against the background of humming computer fans, chatting work colleagues, or the perpetual London-traffic jams outside the window.

All of our senses have this in common: take climbing into a scalding bath which, moments later, feels warm and comforting, or how garlic breath is never noticeable to its owner. Even advertisers have picked up on the idea of ‘sensory adaptation’ by trying to sell us those little plug-in air fresheners that change smell every few days so we never get a chance to get used to them and our noses are perpetually assaulted by a miasma of floral fragrance. While sensory adaptation in humans has been described for more than a century, the ability of single-celled organisms to perform a similar feat is a relatively new finding. However, a group of researchers at the FOM Institute for Atomic and Molecular Physics and the Massachusetts Institute of Technology have been investigating sensory adaptation in Escherichia coli and recently published their finding in the Proceedings of the National Academy of Sciences.

Electron micrograph of microbial cell possessing multiple flagella, Yutaka Tsutsumi

Free-swimming bacteria are propelled by long, whip-like structures called flagella. When the motor controlling the flagella is rotated in a counter-clockwise direction, the flagella bunch together to form a corkscrew that propels the cell forwards. These periods of smooth-swimming are broken up by short bursts of tumbling when the motor is rotated in a clockwise direction, resulting in the flagella turning in opposing directions to re-orientate the bacteria. By varying the time spent swimming and the frequency of tumbles, the cell can effectively direct what is essentially a random process.

The way in which bacteria direct their movement to search out food or to move away from poisons relies on a chemical sensing system in a process known as chemotaxis. Being around two micrometres in length, bacteria act as point-sensors—they simply aren’t long enough to determine how a gradient differs along their length. Instead, they move up or down a gradient by comparing one measurement—a snapshot of the surrounding environment—with the conditions experienced moments earlier. This ability to sense the surroundings from moment to moment and respond accordingly relies on a surprisingly simple system.

The concentration of an attractant is detected by a membrane-bound receptor that transmits this information to the motor controlling the flagella. When no attractant is bound by the receptor, a protein called CheY is activated. In its active state, CheY can interact with the motor, causing it to rotate in a clockwise direction and the cell tumbles. However, upon attractant binding by the receptor, CheY is switched off and the motor turns in a counter-clockwise direction meaning that the cell swims smoothly. If the cell continues to experience increasing concentrations of an attractant, its periods of smooth-swimming become longer. But, if the concentration decreases, CheY again binds the motor and causes the cell to tumble in the hope that it will re-orientate the cell to swim in the right direction.

While this system has already been studied in some detail, the work described in the PNAS paper was interesting because it demonstrated that it is not the magnitude of the change in concentration that matters to the bacteria, but the fold-change. Regardless of the starting concentration, cells respond to a ten-fold increase in attractant in the same manner and this observation holds true over a 10,000-fold range of background concentrations.

The significance of this could have something to do with the extreme variations in nutrient concentrations experienced by bacteria in its natural environment. For example, nutrient patches in the ocean are few and far between and can vary in intensity. If a bacterium was permanently calibrated to search out very high concentrations of nutrient, it could fail to detect a lower concentration. Reaching any source of nutrition, even a small one, could mean the difference between life and death for a cell. Bacteria, it seems, cannot afford to be picky.

How a cell responds to small changes in concentration when there is little attractant present but doesn’t place as much significance on a change of the same magnitude when in a high concentration environment has been difficult to tease apart in the past. One of the difficulties is that, being extremely small, cells are difficult to investigate on the single-cell level. So, traditionally, microbiologists have studied large populations of cells. However, the subtle nuances of a given process can be lost among the noise associated with a large-scale experiment. A technique used in this study, however, partially circumvented this issue.

Microfluidic chemostat - Frederick Balagaddé, California Institute of Technology

Microfluidics involves the use of a miniaturized continuous culture device to investigate tiny volumes under strictly controlled conditions. Generally comprised of a microchannel attached to what looks like a computer chip, a microfluidics system works on an extremely small scale: between 10-9 and 10-18 litres, corresponding to as little as 100 cells. The size of the channel allows automated microscopy by maintaining the cells in a single focal plane. In addition, by labelling the bacteria with either fluorescence of luminescence, the gene expression of single cells can be investigated.

Compared to a large scale chemostat, microfluidics has a number of advantages. One of these is that cells in a microfluidics system tend to be more homogenous-there is less cell to cell variation. In a chemostat, bacteria has a tendency to cling to the walls to form what is known as a biofilm and these surface-attached cells can form a significant proportion of the population within the chemostat. The various sub-populations of cells can skew the results and make subtle changes difficult to elucidate. In addition, chemostats require the cells to undergo numerous divisions which acts to select for spontaneous mutations which lend a fitness advantage. Microfluidics, however, can get past these problems. By looking at very small numbers of cells on a single-cell level, and by reducing the number of cell divisions, even extremely delicate oscillations can be detected. 

The next steps in this area of research is to determine the mechanisms used by the bacterium to adapt to the varying concentrations. Perhaps E. coli possesses more than one receptor for a given attractant, each sensing a different range of concentrations. Or a single receptor may be recalibrated depending on background levels. Microfluidics represents a useful method to study this phenomena further, particularly because it has the potential for investigating the response to a number of opposing attractant and repellant gradients, better representing the environment experienced by bacteria in real life. Not only does this have implications for the understanding of chemotaxis, but could shed light on the mechanisms used by pathogenic bacteria to colonise their hosts.

Link to original article: http://www.pnas.org/content/108/33/13870.long