CH391L/S14/MotilityandTaxis

Introduction
Motility refers to the ability of a biological entity to move spontaneously and actively. Taxis refers to the capacity of an organism, usually unicellular organisms (and simple multicellular organisms), to move upon the presence of an external stimulus (i.e. light, organic and inorganic substances, etc.). The type of taxis that requires the presence of a certain chemical substance is known as chemotaxis. Chemotaxis requires a complex signal transduction pathway with the participation of multiple external receptors (chemoreceptors in the cell membrane) and proteins in charge of controlling the cell motility. This ability of the cell to move upon external stimuli has enabled the synthetic biology vision of exploiting cell motility and taxis for applications such as bioremediation, disease site detection, drug release vehicles, etc. Recently, chemotactic bacteria have inspired surreal research works such as engineering bacteria for cancer cells detection and invasion Adler1974.

Chemotaxis in E. coli
. Chemotaxis was first detected by early microscopes by Leeuwenhoek, the first full descriptions were made by Engelmann (1881) and Pfeffer (1884). This phenomenon was first detected in an experiment were neutrophils were placed in a gradient of fMLP (N-formyl-methionine-leucine-phenylalanine), a peptide chain produced by some bacteria, cells recognized this chemical and migrated towards it.

Chemotaxis in E. coli involves five membrane chemoreceptors and post-transcriptional modifications in six proteins (CheA, CheB, CheR, CheW, CheY, and CheZ) Baker2006  Wadhams2004. This signal transduction pathway allows E. coli to move towards a higher concentration of chemoattractants or away from higher concentrations of chemorepellents. This sensing mechanism allows individual cells to travel to areas more favorable for their survival and growth (Figure 1).

More specifically, these five chemoreceptors in charge of sensing attractants and repellants are sensory histidine kinases in turn coupled to phosphorylable response regulators (CheA, CheB, CheR, CheW, CheY, and CheZ) in charge of controlling the directional rotation of the flagellar motor (Figure 2). In the absence of the chemical gradient the flagellar motor rotates clockwise allowing individual cells to execute random walk. On the other hand, when rotating counterclockwise, the cell performs long runs up a chemoattractand or down a chemorepellent. Once the concentration of the chemical is constant, the cell resumes its random walk movement. An important feature of chemotaxis in E. coli is that individual cell do not only respond to absolute concentrations of a stimulus, they integrate such concentrations over time and respond accordingly based on the concentration differences between two time points.

Engineering the chemotactic sensitivity in E. coli


Goulian and co-workers Derr2006 back in 2006 developed a simple method to obtain variants of chemoreceptor proteins that enable cells to swim towards target attractants. The method is based solely on a diffusive concentration gradient of target attractant in semi-solid agar media (by spreading a thin line of attractant in the center of an agar plate). The researchers focused on the E. coli aspartate receptor tar. They confirmed the relative plasticity of the protein as some of the tar alleles, specifically the variant that responded to cysteic acid still showed strong sensitivity to aspartate. Tar mutants with increased sensitivity to phenylalanine, N-methyl aspartate and glutamate showed a considerable decrease in their response to aspartate.

Grounded on this previous work, Goulian and co-workers continued to develop methods for increasing the chemotactic sensitivity E. coli for novel molecules. Goldberg et al. Goldberg2009 engineered E. coli chemotactic response by synthetically introduce enzymatic activity in the periplasm that converts the target molecule to the native chemoreceptor ligand (Figure 2). This approach was used for the design of two systems. In the first system asparagine responsive strain was engineered by using the E. coli asparaginase II, a hydrolase that degrades asparagine to aspartate. The strain was genetically engineered for the enzyme to be produce at normal conditions and since the strain lacks the chromosomal genes encoding all five chemoreceptors, the receptor Tar was expressed from a plasmid. In the second system, the researchers introduce phenylacetyl glycine (PAG) sensitivity by co-expressing from a plasmid the penicillin acylase (Pac) gene from E. coli strain W (with its native promoter) and the previously selected phenylacetic acid tar receptor (TarPA).

Engineering chemotactic intracellular pathways in E. coli via RNA molecules




Topp and Gallivan Topp2006 demonstrated that by engineering a riboswitch to control E. coli chemotaxis rather than selecting for a chemoreceptor with modified sensitivity could be an efficient method to accomplish novel chemotactic responses. Riboswitches are non-coding RNAs that regulate gene expression via structural reconfiguration upon binding of a ligand. Located at the 5’ untranslated (and les often at the 3’ UTR) of the messenger RNA that they control, these structures are sensitive to ligands, usually small molecules such a metabolite (an amino acid) of the same pathway that they control. The researchers introduced the one of the chemotaxis genes that regulates the directional rotation of the flagellar motor, CheZ, under the control of theophylline-sensitive riboswitch. They sought to bypass the chemoreceptor approach entirely by reprogramming how the intracellular chemotactic pathway works. In E. coli, the 5 different chemoreceptors are linked to the chemotaxis proteins CheB, R and W, which collectively control CheA phosphorylation. CheA phosphorylates CheY that in turn controls the rotational direction of the flagellar motor by binding to the flagellar switch protein FliM. When CheY is phospholyrated the flagellar motor rotates clockwise, which causes the cell to tumble. In contrast, when CheY is dephosphorylated by CheZ, the flagellar motor changes direction to counterclockwise rotation allowing E. coli to execute smooth swims. E, coli lacking the CheZ gene stumbles permanently, thus becoming non-motile. It has been demonstrated previously that inducing CheZ expression restores chemotaxis in a CheZ knockout (Figure 3).

The authors accomplished a process called pseudotaxis by controlling CheZ with a theophylline-sensitive riboswitch. Pseudotaxis differs from chemotaxis in that cell motility is dictated by the absolute theophylline concentration as opposed to changes in concentration over time. Figure 4 shows not only that the authors accomplished pseudotaxis triggered by theophylline absolute concentration but also that the cell migration can be guided across a specific path on a semisolid media plate.

Control of E. coli cell localization within a consortium via the artificial manipulation of chemotaxis


In a representative example, Goldberg et al. Goldberg2009 in the same study described in section above exploited the “hitchhiker” effect observed in which cells lacking the enzymatic activity can be induced to motility by cells that produce the proper ligand. They engineered two E. coli strains to form a consortium in which each of them can produce the ligand that induces chemotaxis in the other strain. Specifically, one strain was engineered to produce the native aspartate chemoreceptor and penicillin acylase, the other strain was designed to express a PAA-responsive mutant chemoreceptor and asparaginase II. The observed behavior of the consortium was that of an “AND” Boolean gate since when each strain was isolated and put in contact with the appropriate ligands, no chemotaxis was observed. In contrast, when the two strains were plated in close proximity, the two chemical signals were present and the strains were motile (Figure 5). This research work advances the possibility of utilizing these engineered strains in a real environment since bacterial populations are usually better fit to thrive when living in consortia.

Engineering the intracellular chemotactic pathway vs the manipulation of chemoreceptor sensitivity
The advantages and disadvantages of both approaches presented above are summarized in the following table (information taken from Mishler2010 ):

Potential applications: the paradoxic “therapeutic bacterium” and more
In a proof of concept of a medical application, Anderson et al. Anderson2006 engineered E. coli to invade cancer cells depending on the local environment. The researchers control E. coli behavior by introducing the gene that allows cell invasion, inv gene, under the control of native promoters that act under specific conditions of hypoxia or cell density. By this approach they were able to control when and where cell invasion take place. This example and the others described above enable the vision of almost surreal applications such as a “therapeutic E. coli” in which its chemoctactic activity is engineered to detect disease sites and then release drugs for whose production the organism had been rewired a priori. This means that the field of potential applications is unlimited in areas such as target cell therapy and regenerative medicine, drug delivery, and even other non-medical applications such as biosafety and anti-biofouling in pipes, boats, implantable devices and surgical instruments Vazquez2013.

Engineering other types of motility
Synthetic Cilia-like structure engineered to help understand the functioning of biological cilia

Cilia are highly conserved eukaryotic structures essential for reproduction and survival of many biological organisms. In this work, Sanchez and collaborators Sanchez2011, molecularly engineered cilia to study how the biological version functions. The authors describe a minimal model system composed by densely packed, actively bending bundles of microtubules and molecular motors, spontaneously synchronize their beating patterns. This simplified in vitro model of eukaryotic cilia could bring insights into the beating of isolated cilia and the synchronization of their beatings.

iGEM projects on chemotaxis
A couple of examples of iGEM projects associated with chemotaxis and motility engineering are as follows:

Back in 2012, team Göttingen developed a selection method to engineer a strain that they called “Homing E. coli”. Through genetic manipulation and directed mutagenesis, they were able to optimize E. Coli’s capacity to quickly travel through a chemo-attractive gradient of aspartate. They identified FlhDC as a cellular factor for flagella enhancement and elongation, and created a library of receptors that they will soon explore to modify specificity of the receptor and, ultimately, motility of the E. Coli. Specifically they applied directed evolution to chemoreceptors by targeting five amino acid residues in the ligand binding site. In order to select for the appropriate phenotype, a new special "swimming" plate was developed by using a low concentration of agar (0.3%). Several tests were run to determine incubation times and temperature, nutrients and chemoattractant concentrations, and even the right strain. They chose BL21 cells in 0.3% swimming agar at 33 C for 1-2 days. After determining the appropriate "swimming" assay, the team started investigating genes involved in the flagellar function of E. coli and their effect on bacterial motility when over-express from a plasmid. They found that gene yhjH had a relatively important positive effect in the "swimming" behavior of the cells since by over-expressing from plasmids, the ability of cells to brake was considerably diminished and biofilm formation appears to repressed. These results are in accordance to the functions that gene yhjH regulates in E. coli. fliC (flagellin) encodes for the structural protein that builds up the filament of the bacterial flagellum. By overexpressing it the authors observed a similar behavior to yhjH versions. A higher motility than the control was observed in general when overexpressing flhDC (master regulator of motility and chemotaxis), however inconsistent results prevent the authors from obtaining definitive conclusions. In general, by overexpressing genes such as yhjH (releases brake and spreads individual cells), fliC (makes stronger and elongated flagella) and flhDC (increases number of flagella), a relatively positive effect on E. coli motility was observed iGEMGottingen2012.

In 2010, team UPO-Sevilla explored their capacity to concentrate populations of bacteria, or enhance a phenomena termed “Bacterial Crowding”. This fundamentally requires a strain (containing the Prh system) to sense a non-diffusive chemical attractant and subsequently release signals to attract other bacteria to crowd there. This team used two chemo-attractants: aspartate and salicylate, in the hope of demonstrating that they can induce crowding of a strain that is not chemo-sensitive to salicylate.