Team:FSU/Design
OVERVIEW
Our first system involves using sound as a mechanical force to open a mechanosensitive channel within the E. Coli membrane. This opening allows the diffusion of ions into the cell. Zinc was an ideal choice for our mechanosensitive system due to it slow exchange between its extracellular pool and intracellular binding, in a low concentration. ZntR is a zinc-dependent transcription factor that binds to the promoter upstream of zntA in the E. Coli genome. Zinc is naturally exported by E. Coli and ZntA is an active transport system for the export of zinc. Zinc-bound zntR will bind to our promoter sequence upstream of mRFP. ZntR then transribes zntA to export zinc out of the cell. This will demonstrate that zinc successfully entered the cell via sound and activated a genetic response.
Our second system is the direct protein-directed detection of sound stress. RpoE, the sigma 24 factor of RNA polymerase, is known for its response to stress that affects outer membrane proteins and membranous lipopolysaccharides. While the signal cascade to activate RpoE is induced by many stress-sensing proteins, we only require one such protein to "detect" sound. An activated RpoE will upregulate expression of BamE, which is responsible for outer membrane protein aggregation and membrane permeability.
BamE is located on the outer membrane and once activated by RpoE, it very likely responds to stress on the membrane by repairing damaged proteins. The viability of this system can be evaluated by striking it with sound, and then examining the membrane for it's repaired structure after a period of time.
The Rcs "Regulator Capsule Synthesis", our third system, is a two-component signal transduction system that regulates critical cellular functions in response to changing environment. The regulated functions include cell division, activity of periplasmic proteins, motility, biofilm formation, etc. Protein RcsC acts as a sensor kinase that autophosphorylates in response to an environmental signal such as changing osmolarity, or the expression of an outer membrane protein, and then transfers the phosphate group to inner membrane protein RcsD, and then to the cytosolic response regulator RcsB.
RcsB can transcriptionally activate genes that regulate the synthesis of periplasmic proteins. One such is OsmC, an osmotically-inducible peroxiredoxin. Expression of OsmC is also transcriptionally activated by NhaR, which is necessary for this gene's activation in the presence of NaCl, LiCl, and sucrose (Toesca et al., 2001). If this genetic system successfully responds to sound, measurable expression of OsmC should result.
The pspABCE operon of the "Phage Shock Protein" represents our fourth system, which responds to membrane stresses. Under normal cell conditions PspA suppresses PspF. During membrane damage and loss of proton motive force the pspBCE cascade suppresses pspA thereby freeing PspF. PspF is then free to bind upregulate several genes including the pspABCE operon. Sound stress may be able to damage the bacterial membrane or disrupt the proton motive force.
PspA, while serving as a proteinaceous response to membrane damage, may have a unique response to sound that involves the activity of mechanosensitive channels. If sound disrupts the membrane and thereby damages the proton motive force, simultaneous Msc channel activation may re-allow the protons back in, as PspA functions to restore the proton motive force and expel the protons.
Design Principles
Sound as Stress
The plasma membrane is the interface for conversion of environmental stimuli into biochemical signals that induce a genetic response within the cell. While there are few studies that focus on sound as an environmental stimulus on bacteria, there is a plethora of evidence for similar stimuli, such as osmotic shock, heat shock, and metal ion exposure. We hypothesize that sound may induce a similar "shock" response on the membrane. Osmotic shock, for example, can create turgor pressure upon the plasma membrane when extracellular solute concentration is low, so we hypothesize that sound can cause a similar pressure on the plasma membrane, thus resulting in stress.
Mechanosensitive Channels for Transduction of Sound
Since sound is ultimately a wave of pressure, it can be considered to be a mechanical stress. Within the plasma membrane of E. Coli cells exist various mechanosensitive protein channels, whose primary role are to protect the cell's integrity from shifts in osmolarity. The most common "Msc" channels in E. Coli are MscM (mini-conductance), MscS (small conductance), and MscL (large conductance). They respond to mechanical stimuli by transmitting ions and electric flux via a gate-opening mechanism, changing the cell's membrane potential. Mechanical stimuli include forces that create changes in the tension of the lipid bilayer of the plasma membrane, created by a deformation affecting the membrane curvature and inducing a bilayer-protein hydrophobic mismatch. We hypothesize that sound exertion can create such a tension, forcing the channel from a closed state to an open state.
The mechanosensitive response
Our Sound-Induced Systems
Two types of systems were tested in our experiments. The first type of system utilized ions as ligands whose signaling was activated by sound; and the second system involved a membrane stress response, in which the cell contains pre-existing protein systems that respond to external stress on the outer membrane. In this case, the stress is sound. For our ion ligand system, we were able to select ions based on factors that would induce a mechanoreceptive response. Bacteria are generally non-selective in their signaling molecules, so long as they are within a certain atomic radius. Therefore, we searched for ions that tend to be exported from the cell and that have a relatively low intracellular concentration. With this tendency, sound could be utilized to open a channel and "pull" the ions back into the cell. Our second system involved testing the response of stress-induced protein systems to sound. The functions of these proteins, such as RpoE, PspA, and RcsB, are to protect the cell from damage by the stress.
To be added:
https://ecocyc.org/