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 transmission of our zinc ion signal to flow into the cell, activating our genetic response system. Upon entering the cell, zinc will bind to ZntR, a zinc-dependent transcription factor. Active ZntR will bind to a promoter sequence upstream of the gene zntA, coding for a zinc exporter protein channel.
Our second system involves a direct 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. RpoE can become active due to the activity of many stress detecting proteins, we only require one such protein to "detect" sound. An activated RpoE will upregulate expression of BamE. BamE is responsible for outer membrane protein aggregation and membrane permeability.
The Rcs "Regulator Capsule Synthesis" system is a two-component signal transduction system that regulates critical cellular functions in response to changing environment, such as membrane damage. The regulated functions include cell division, activity of periplasmic proteins, motility, biofilm formation, etc. Protein RcsC autophosphorylates in response to an environmental signal and then transfers the phosphate group to inner membrane protein RcsD, and then to the cytosolic response regulator RcsB. RcsB can transcriptionally activate OsmC, an osmotically-inducible peroxiredoxin. Expression of OsmC is also transcriptionally activated by NhaR, the regulator of the sodium-hydrogen antiporter.
The Psp "Phage Shock Protein" system consists of the pspABCDE operon, which responds to membrane stresses and is activated by PspF. Under membrane disruption, PspA maintains the proton motive force by suppressing it from extruding hydrogen protons through a disrupted cell membrane caused by cellular stress, such as sound.
Design Principles
Sound as Stress
The plasma membrane is the platform 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, 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 is to protect the cell integrity from osmolarity transition. 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 changes in the tension of the lipid bilayer of the plasma membrane, created by a deformation of the lipid membrane that affects the membrane curvature and induces 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 general structure of the homoheptameric MscS channel consists of: three transmembrane helices, a C-terminus facing the selective and stable cytosolic domain and an N-terminus facing the periplasmic domain. Transmembrane 1 (TM1) and TM2 form a flexible paddle for tension-sensing, while the conserved TM3 forms a linked amphipathic helical pore structure. A channel pore connects the periplasm to the cytosolic vestibule, and a selectivity filter exists in the middle of the channel. The pore has a diameter for conductance values ranging from 14-16 amps, and opens and closes in a force-activated gating manner.
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 between the signaling molecules they select, 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. Zinc was an ideal choice for this criteria, in addition to the fact that it has a slow exchange between its extracellular pool and intracellular binding. Once zinc enters the cytosol, ZntR 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 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. 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.
RcsB is activated by the two-component sensor kinase/response regulator signaling system of RcsC and RcsD. RcsC responds to signals such as changing osmolarity, or expression of an outer membrane protein. Once RcsB receives the phosphorylation signal from RcsD, it can transcriptionally activate genes that regulate the synthesis of periplasmic proteins. If the Rcs system successfully responds to sound, measurable expression of OsmC should result.
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.
To be added:
https://ecocyc.org/