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Introduction

The Effects of Sound on Bacteria

Investigators report that the growth rate of bacteria increases when exposed to audible sound (Lee Ying, Dayou and Phin, 2009). Increased E. Coli growth rates were observed when cultures were exposed to 5 kHz sound (Lee Ying, Dayou and Phin, 2009). Other studies indicate that E. Coli exposed to a wide range of frequencies, 500 Hz to 16 kHz, generally showed an increased biomass, in particular, 8 kHz was found to increase protein and RNA concentration (Gu, Zhang and Wu, 2016). The investigators reported a positive correlation between sound power, measured in decibels, and bacterial biomass (Gu, Zhang and Wu, 2016). Other studies report that bacteria generate sounds that may enhance the growth rates of neighboring bacteria (Matsuhashi et al., 1998). The investigators found that frequencies of 6-10, 18-22, and 28-38kHz promoted B. Carboniphilus growth rates (Matsuhashi et al., 1998). They also found that B. subtilis emits sound between 8 kHz and 43 kHz, with the most intense waves at 8.5, 19, 29, and 37 kHz. The studies we reviewed point to the possibility that E. coli could be induced to express a gene using sound. Our project pursues this possibility.

2008 Berkeley iGEM Team

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. Since sound is ultimately a wave of pressure, it can be considered to be a mechanical force that can create a local stress, such as membrane tension. We hypothesize sound may cause membrane perturbations, altered membrane tension, protein misfolding, and increased growth rates. Membrane perturbations are tested using the pspA system. Increased membrane tension is tested using the zntA system. Protein misfolding is explored with bamE design. The osmC system tests the upregulation of cell surface proteins, cell growth pathways, and protection proteins against a changing environment.

Mechanosensitive Channels for Transduction of Sound

Within the double membrane of E. Coli cells exist various mechanosensitive protein channels, whose primary role are to protect the cell's integrity from shifts in osmolarity. For example, if there are too many solutes in the cell's cytoplasm, water will flow into the cytoplasm to lower the osmolarity, creating a turgor pressure that pushes outward against the inside of the plasma membrane; once a certain pressure is reached, the MS channels will open (Booth, 2014).The channels respond to mechanical stimuli by conformational change; a gate-opening mechanism then allows the transmitting ions and electric flux, changing the cell's membrane potential (Martinac, Adler and Kung, 1990). 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 (Malcolm, Blount and Maurer, 2015). It has been shown in previous iGEM experiments that the MscS can be opened using ultrasound (Leben et al., 2016). Previous iGEM experiments with eukaryotic mechanosensitive channels, TRPC5 and Piezo1, have shown sound-mediated opening (Fan, J et al., 2016).

Our Sound-Induced Systems

Two general systems were employed in our experiments.

The first utilized ions as ligands activating a sensing mechanism within the cytoplasm. When the ions' intracellular concentration increased, so would the bacterial pathway. We searched for ions that tend to be exported from the cell and that have a relatively low intracellular concentration. When a mechanosensitive channel opens the ions are able to diffuse quickly down their concentration gradient. Two component systems, designed to detect a certain ion, is utilized to detect this influx.

The second system involves membrane stress response pathways. Sound stress could trigger pathways important for adapting to the environment and protecting membrane integrity. This system does not rely on the addition of any substances to a bacteria culture, only sound frequencies. Sound can potentially cause significant stress to the E. Coli membrane and membrane proteins.

OVERVIEW

zntA System

Our first design 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. zntR is a zinc-dependent transcription factor and will detect the influx of zinc ions into the cell cytoplasm. Zinc was an ideal choice for our mechanosensitive system due to its slow exchange between its extracellular pool and low cytosolic concentration, with a tendency to be extruded from the cell (F C Kung, 2018). Our plasmid incorporated the promoter for zntA, an ATPase zinc exporter, keeping intracellular zinc at a minimal concentration of 0.2 mM (Outten, Outten and O'Halloran, 1999). msc opening due to sound pressure will be indicated using an mRFP signal. mRFP is incorperated into our plasmid downstream of the zntA promoter.

zntA Pathway Mechanism

Sound, acting as a mechanical pressure wave, has the capacity to open mechanosensitive channels. The opening of an msc will allow in surrounding molecules including zinc ions. zntR is an E Coli zinc detection protein. zntR requires the binding of 4 zinc ions before it can be an active transcription factor. The zinc-zntR complex has an affinity for the binding site at -35 and -10 upstream of the zntA promoter.

Here, ZntR will alter the zntAp DNA structure to make it a better substrate for RNA Polymerase, thus activating transcription of zntA (Outten, Outten and O'Halloran, 1999). zntA will act to export zinc from the cell and turn off the ligand signal for zntR. ZntA will then export cytosolic zinc out of the cell using a ATP hydrolysis dependent mechanism: one zinc ion removed for every ATP hydrolized (Rensing, Mitra and Rosen, 1997).In our plasmid we have mRFP downstream of the zntAp, thus mRFP will be produced in response to this system. Because our plasmid is high copy number it is less likely that zntR will be able to find the genomic zntAp copy. Thus, the export of zinc will be minimal.

Mechanosensitive Channels

Other ionic mechanotransduction systems exist in E. Coli that are similar to our zntA design. Unlike most eukaryotic channels, bacterial MscS and MscL are unbiased in their selection of molecules and ions. MscS and MscL allow in molecules up to 1,000 M.W. (Peyronnet et al., 2014). This implies that a variety of signaling ions and macromolecules can be transduced through mechanosensitive channels. The most common "Msc" channels in E. Coli are MscM (mini-conductance), MscS (small conductance), and MscL (large conductance). Calcium is known to iGEM to travel through eukaryotic mechanosensitive channels: Piezo1 and TRPC5 (Fan et al., 2016), and E. Coli MscS (Leben et al., 2016) upon opening with sound pressure. It is therefore very likely that zinc will also travel through the MscS and/or other msc channels. Other E. Coli mechanosensitive channels include MscK, MscM, YnaI, YbiO, YbdG.

Compared to other ions, zinc exhibits an ideal physiological nature for our system. Calcium and magnesium are well-known signaling ions; however, calcium signaling is often inhibited by zinc and magnesium ions. Furthermore, magnesium has a better binding affinity for oxygen and is more often bound as a cofactor (Chaigne-Delalande and Lenardo, 2014). Magnesium, potassium, and sodium also exhibit a smaller chemical gradient across the plasma membrane than zinc does, and therefore they have a tendency to be imported into the cell (F C Kung, 2018). And unlike other transition metals that are bound as cofactors, zinc is often ionized in physiological solutions, implying a regulatory role in ion channels (Elinder and rhem, 2003).

rpoE System

Our second system involves the protein-directed detection of sound stress. RpoE, the sigma 24 factor of RNA polymerase, is known for its response to stress on outer membrane proteins (OMPs) and membranous lipopolysaccharides (LPS). Such stresses include misfolding of OMPs, heat shock, osmotic shock, and change in membranous LPS structure (Rouvière et al., 1995). A protein-based signal cascade activates RpoE's transcription, allowing it to activate a number of stress-response genes. RpoE can upregulate BamE, which is an OMP that is responsible for maintenance of OMP aggregation and membrane permeability (Rigel et al., 2011). For our system a sound-activated RpoE will bind to bamEp within a region of -41 b.p. before the transcription start site to upregulate mRFP.

RpoE Pathway Mechanism

To activate RpoE in our system, sound stress must strike the E. Coli cell culture and denature an OMP. A misfolded OMP binds to the protease DegS in the periplasmic space. DegS is then able to cleave RseA/B, which physically tethers RpoE to the inner plasma membrane. Once RpoE is released, it is free to bind RNA Polymerase and form the RNA Polymerase Holoenzyme. The addition of RpoE to RNA Polymerase is essential for the upregulation many genes, including its own and bamE. In our design we use the bamE promoter to upregulate mRFP. A byproduct of our system is BamE, which is a repair enzyme for the outer membrane. A positive feedback loop increases the concentration of RpoE until the stress is reduced and degradation of RseA slows down (Ades, Grigorova and Gross, 2003).

osmC System

Our particularly advantageous third system involves the promoter OsmCp1 of the cytosolic peroxiredoxin, OsmC, that is stress-activated by two different transcription factors that are independent of each other: RcsB and NhaR. OsmC expression is induced under osmotic pressure (Toesca et al., 2001), a mechanical force similar to sound; its function is to defend against the oxidative stress of organic hydroperoxides (Lesniak, Barton and Nikolov, 2003). OsmCp2 is another promoter of the OsmC system and is also induced by osmolarity, but it is activated upon entry into the stationary phase and transcribed by RpoS. We have incorporated the mRFP DNA sequence upstream of OsmCp1 in our plasmid, hoping for a twice as likely chance of activation, as well as the possible byproduct of OsmC from the E. Coli genome.

Rcs Pathway Mechanism

The Rcs "Regulator Capsule Synthesis" system is a two-component signal transduction system that regulates critical cellular functions in response to environmental effects on the cell envelope. Originally known for regulating capsular polysaccharide synthesis, other regulated functions include cell division, activity of periplasmic proteins (i.e. OsmC), motility, biofilm formation, etc. RcsF is an OMP that activates RcsC for the signal transduction by removing the inhibitory effect of IgaA on the Rcs system. Protein RcsC acts as a sensor kinase whose histidine domain autophosphorylates in response to an environmental signal such as the alteration of the outer membrane proteins, and then transfers the phosphate group in a conserved manner to inner membrane phosphotransferprotein RcsD, and then to the aspartate residue of the cytosolic response regulator RcsB (Davalos-Garcia et al., 2001). Once RcsB is phosphorylated, its transcriptional capability is enhanced and it can bind to OsmCp1.

NhaR Pathway Mechanism

NhaR, while independent of RcsB, is necessary and powerful inducer for OsmCp1's activation in response to osmotic shock. More specifically, NhaR transcribes OsmCp1 in the presence NaCl, LiCl, and sucrose by binding to one of these solutes in the cytosol and then to the promoter sequence (Toesca et al., 2001). Furthermore, increased ionic osmolyte concentration has been shown to stimulate induction of OsmCp1 (Toesca et al., 2001). If either NhaR or Rcs activate OsmCp1 in response to sound, measurable expression of mRFP should result. We selected a promoter region of -191 b.p. before the T.S.S. of the osmC gene for our system; and in the E. Coli genome, the sequence that's necessary for NhaR activation is adjacent to the -35 OsmCp1 region.

pspA System

The pspABCE operon of the "Phage Shock Protein" represents our fourth system, which responds to membrane stresses that deplete energy and uniquely utilizes a proteinaceous stress response as well as an ion signal. This includes phage infection, osmotic shock, exposure to hydrophobic organic solvents, blockage of Sec machinery for protein export, etc.(Brissette et al., 1991). PspA of the inner membrane specifically responds to loss of the proton motive force (PMF) by suppressing proton leakage of damaged membranous phospholipids and of damaged liposomes. Loss of the PMF can be caused by a force opening an MscS channel, resulting in an influx of protons. If sound disrupts the membrane and PMF, while simultaneous Msc channel activation the protons back in, PspA can function to restore the proton motive force and expel the protons. Therefore for our system, we selected promoter sequence pspAp within -142 b.p. before the T.S.S. for pspA; naturally, bound PspF (the transcriptional activator for pspABCE) binds between -127 and -111 b.p. before the T.S.S. Our mRFP sequence will be located upstream of pspAp.

PspA Pathway Mechanism

Under normal cell conditions PspA suppresses its own expression by inhibiting PspF. During membrane damage and loss of PMF, the pspBCE cascade suppresses pspA thereby freeing PspF. PspF is then free to bind upregulate several genes including the pspABCE operon and the promoter, pspAp (Jovanovic et al., 2006). PspB and PspC are utilized to activate the psp operon. Sound stress may be able to damage the bacterial membrane or disrupt the proton motive force, to which PspA may respond to, if transcribed successfully for the E. Coli genome.

Plasmid Construction

Each plasmid was constructed using two gBlocks to form the insert and a chloramphenicol backbone. The first gBlock contains a biobrick prefix, the putative promotor, and the first 25 base pairs of the mRFP1 gene sequence. The second gBlock contains the full mRFP1 gene sequence, the BBa_B0015 Double Terminator, and the biobrick suffix. Using the New England Biolab (NEB) Hifi DNA Assembly Master Mix protocols, the biobrick prefix and suffix allowed one end of both gBlocks to anneal to their respective sides of the Chloramphenicol backbone and the mRFP1 gene to anneal the two gBlock segments together. This process finalized the construction of our test devices and were then transformed into NEB 5-alpha cells for testing.

BIOFAB Collection

In order to mitigate the risk of the sound inducible project we decided to add constitutive promoters from the BIOFAB project. This project’s goal was to create a library of promoters that would allow for consistency in the synthetic biology community, we took these parts and made them available to the iGEM community. We then tested three promoters apFAB46, apFAB82, and apFAB90 from this collection with high, medium and low activities, respectively.

The plasmid maps shown describe the design of our plasmids for the BIOFAB test devices. Testing these promoters consisted of adding the BIOFAB promoter, shown in bright green, to RFP, shown in blue, to a chloramphenicol resistant plasmid. These plasmids were transformed to the NEB-5-alpha and grown to stationary phase. We used a spectrophotometer to test fluorescence at OD/700 and compared them to a negative control with no RFP and a positive control with the iGEM RFP control BBa_J04450.

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