Fine-tuning of chemotactic response in E. coli determined by high-throughput capillary assay.
Wiki by Bryan Kaye
Title: Fine-Tuning of Chemotactic Response in E. coli Determined by High-Throughput Capillary Assay
Authors: Heungwon Park • Calin C. Guet • Thierry Emonet • Philippe Cluzel
E. coli search for food and avoid toxins by examining concentrations of chemical markers (aka Chemotaxis). The algorithm that E coli use to find food has been examined by swarming arrays. A swarming assay is usually made by placing a layer of agarose gel between the bacteria population and a chemoattractant. As the chemoattractant diffuses through the agar layer, researchers can watch how the bacteria move in response. Studying chemotactic response is assays are difficult because of cellular growth and depletion of nutrients.
In addition to a swarming assay, Park et al. use a high throughput capillary assay that eliminates the effects of cell growth. They measure both the chemotactic response to L-aspartate and the concentrations of YFP-CheR and CFP-CheB (florescent proteins made in response to the cell’s detection of nutrients that cause changes in cell mobility). They found that the chemotactic response was tailored to a specific ratio of CheR:CheB. Therefore, the response was not adaptive.
Bacteria move by using many rotary motor flagella positioned at the cell membrane. The flagella are in sync when all of them are rotating counter-clockwise and the net effect is that the bacteria swims smoothly. This motion is called a run. After a smooth run, if even a few flagella rotate counter clockwise, the bacteria will tumble and orient itself in a seemingly random direction. The bacteria may then go into a run. A sequence of runs and tumbles is described by a random walk. E coli takes advantage of this by lowering the frequency of tumbles in the presence of nutrients, therefore biasing the random walk and giving rise to chemotaxis.
While E coli has perfect adaption of chemotaxis, it is unclear if the internal chemistry of the chemotactic response itself is robust to varying concentrations of CheB and CheR. The authors used many parallel capillary assays to obtain enough data so that the results can be statistically significant. They also used fusion proteins, CheB and CheR with florescent labels, CFP and YFP, respectively, to estimate concentrations of CheR and CheB in the cell under observation.
Materials and Methods
In the swarming assay, chemotactic efficiency is defined as the ratio of the ratio of the diameters of the swarming efficiency of mutant cells to wild type (wild type means the phenotype of species found in nature). The capillary assay is built from 96 narrow needles that are connected to 96-well pipetting device. Half of the needles are controls (contain same medium as wells) and the other half contain 10-2 M of L-aspartate (attractant). A syringe is connected to the needles and is the source of the attractant solution. The attractant is initially coming from the needles that are located 3.5mm from the bottom of the well. The attractant diffuses through the needle and the authors study how well the cells navigate into and up the needle vs those in the control tests. Chemotactic response is defined as number of cells in the capillary (needle) with the attractant to the number in the capillary (control needle) without the attractant. See Figure 1 of article.
Results and Discussion
Swarming Experiments: In mutant cells that had CheR less than wild type, the chemotactic efficiency decreased sharply with CheR. In mutant cells with CheR greater than wild type, there was no difference in chemotactic efficiency between mutant and wild type. In fact, when CheR in mutants was 5 times higher than wild type, chemotactic response was slightly less in the mutant than the wild type.
Capillary Experiments: When CheR and CheB were varied independently in mutant cells, it was found that the best chemotactic responses were comparable to wild type. In double mutants, the maximal chemotactic response was seen where CheR/CheB ~1. The maximal chemotactic response in the double mutants was comparable to wild type.
The chemotactic response from capillary assays peaked more sharply than those of the swarming assays. Cells in the capillary assay were more fine-tuned to the wild-type CheR/CheB ratio. This study showed that chemotactic response of a population of bacteria is fined tuned and depends very heavily on the ratio of CheR:CheB. There is a biochemical negative feedback loop on CheB and this study suggests that this feedback loop may be the result of strong evolutionary pressure for bacteria to have efficient chemotactic behavior.