We thank members of the Ajo-Franklin lab for insightful discussions on BES assembly, maintenance, critical steps, and troubleshooting. The research was sponsored by the Army Research Office and was accomplished under Grant Number W911NF-22-1-0239 (to C. M. A-F, supporting R. A.) and by the Cancer Prevention and Research Institute of Texas, grant # RR190063 (to C. M. A-F, supporting R. C., S. L., and B. B. K.). Figure 1 was created with BioRender.com.

Chronoamperometry Analysis of EET in Lactiplantibacillus plantarum with DHNA

The authors have no competing interests to declare.

Using the three-electrode, two-chamber bioelectrochemical system described here, we showed measurement of current generation from DHNA-mediated EET in L. plantarum. These BES experiments generate high-quality data; however, BESs are sensitive. Thus, protocol success depends on user precision, particularly in reactor and reference electrode assembly, positioning of needles and electrodes within the anodic chamber, and cation exchange membrane replacement. It is critical to assemble reactors carefully, ensuring no water/media leakage during autoclaving or experimentation. Water leakage can be resolved by ensuring cation exchange membranes are cut to precisely fit the O-ring and tightening the knuckle clamp to finger tight. It is also essential to fully submerge the carbon felt round fully in water during autoclaving to allow it to become hydrophilic for experimentation. We recommend new users allow newly assembled reactors filled with water to sit for 2 h prior to autoclaving, checking for signs of slow leaks below the main bottle junctions. Moreover, ensuring a proper reference electrode assembly guarantees consistent data replication across reactors. If the Teflon frit inside the glass housing becomes discolored, cracked, or dried, this can cause a high resistance of the reference electrode. Users can replace the glass housing to restore reference electrode performance.
Proper orientation of all needles and electrodes within the anodic chamber during experimentation is critical to experiment success. The reference electrode must not directly contact any part of the carbon-felt working electrode. Users can adjust the carbon felt position by gently rotating the working electrode titanium wire from above the reactor. Additionally, needle placement for nitrogen sparging should not make direct contact with electrodes within the chamber or any electrode/potentiostat connections above the chamber. The nitrogen stream should be adjusted to not flow into either electrode. Finally, users should make sure the stir bar does not contact the working electrode by positioning the working electrode 1-2 cm above the stir bar. If an erratic signal is observed in OCV, this can usually be resolved by ensuring proper placement of electrodes and the nitrogen stream within the reactor, and by checking that connections between the potentiostat leads and the reactor electrodes are correct and secure. Lastly, our experience shows that electron mediators like DHNA can be retained within the cation exchange membrane and cause a high background current if reused too many times. We recommend replacing the cation exchange membrane after two to three uses, especially when investigating mediated EET, to guarantee reliable experimental results.
Unlike direct EET, where direct microbial attachment to the electrode facilitates electron transfer, mediated EET necessitates consistent diffusion of electron shuttles across the cell membrane and the electrode, resulting in the unique BES settings described here. First, we chose a double-chamber BES over the single-chamber counterpart in our protocol to separate anodic and cathodic reactions with a cation exchange membrane. This separation prevents the freely diffusing electron mediators (DHNA) and microbes from cross-interacting with the cathode, ensuring microbial EET is the major electron source to reduce the electron mediators and the anode. The separation also allows precise control over parameters like mediator concentration/distribution and the potential poised to the anode. Additionally, we chose carbon felt as the anode material amongst other options such as graphite rods, metal electrodes, glassy carbon, or indium tin oxide (ITO). This is because the 3D porous structure of carbon felt provides a much larger surface area than those electrodes30, allowing efficient utilization of mediators even at high concentrations. Our three-electrode, two-chamber BES settings provide a reliable and reproducible readout of mediated EET even over long-term monitoring; however, this process is relatively low throughput. This protocol is suitable for bench-scale understanding of EET mechanisms or to test prototype EET applications. Alternative BES architectures such as portable or printed BESs31,32, complementary metal oxide semiconductor (CMOS) arrays33, or up-scaled BESs34 can be considered by researchers for different fundamental or application purposes.
In this protocol, we provide detailed instructions for the most commonly used electrochemical techniques: chronoamperometry (CA) and cyclic voltammetry (CV). It is worth noting that other electrochemical techniques, such as Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV), can provide deeper insights into the BES by analyzing charge transfer resistance and double-layer capacitance35,36,37. While this BES protocol enables EET measurements, complementing electrochemical data with metabolic activity and cell biomass measurements can also be essential for a comprehensive analysis. Microbes like L. plantarum engage EET as one of the electron sinks alongside other fermentation byproducts such as lactate and ethanol. Moreover, it is noteworthy that cell biomass growth also serves as an electron sink13. Therefore, quantifying consumed electron donors (e.g., mannitol), assessing cell biomass growth, and monitoring fermentation byproducts offer deeper insights into the efficiency and physiological ramifications of EET. Cellular metabolites are commonly quantified using chromatography and enzymatic assays, while cell viability and growth are assessed by counting colony-forming units and measuring the optical density of spent media at 600 nm, respectively13. It is also important to note that EET measurements are sensitive to small perturbations in experimental conditions. This includes but is not limited to pH, temperature, stirring speed, and nitrogen gas sparging rate38. Therefore, normalizing measured EET levels with bioanalytical measurements acts as an internal control, facilitating consistent assessment across experiments conducted on different days.
Combining electrochemical techniques with other bioanalytical measurements, mediated EET creates new opportunities for electro-fermentation and bioelectrocatalysis. Conventional use of organic, inorganic, or enzymatic electrocatalysts poses challenges due to their high cost and are prone to degradation. Alternatively, using microbes as living electrocatalysts offers a less expensive and more scalable solution due to microbes&#39; self-repair and self-replication abilities39. L. plantarum, generally recognized as a safe&#160;lactic acid bacterium, is a particularly intriguing chassis. Using identical electrochemical setups described in this protocol, we have previously shown that L. plantarum can ferment kale juice under EET conditions and accelerate the metabolic flux toward producing more fermentation end-products such as lactate, acetate, and succinate13; these organic acids are essential flavor compounds in food fermentation. This implies that, by using electrochemical techniques, mediated EET in L. plantarum can be potentially hijacked to manipulate metabolic flux, alter food flavors, or produce valuable chemicals. It is worth noting that the electrochemical techniques presented in this protocol can not only be applied to L. plantarum but can also be generically applied to other native or engineered microbes that perform mediated EET40,41. Different electron mediators, such as flavin, ferrocene, neutral red, ferricyanide, lawsone, and menadione can be selected based on the electron transfer mechanism of the specific microbe being used22,42. Moreover, the BES protocol established in this work can be extended to exoelectrogens that perform mediatorless EET as previously demonstrated with Shewanella and Geobacter species43,44. An optimized growth medium should be used to support the cellular activity of the particular microbe to facilitate its EET performance. This protocol fine-tunes parameters for DHNA-mediated EET in L. plantarum, but modifications are expected when a different microbe and electron mediators are applied.

Bioelectrochemical systems interface microbes with electrodes, allowing investigation of extracellular electron transfer (EET) mechanisms and providing renewable approaches to bioelectrocatalysis1,2,3. Microbes that naturally perform EET are known as exoelectrogens, which transfer electrons derived from metabolism to extracellular terminal electron acceptors, e.g., iron (hydr)oxides and electrodes1. First characterized in Geobacter and Shewanella species4,5, EET pathways have since been identified in many&#160;bacteria. These exoelectrogens play a central role in several microbial electrochemical technologies, such as generating electrical energy from waste streams, fixing CO2, and producing valuable chemicals via electrosynthesis1,6,7,8,9,10,11,12.
One such exoelectrogen is Lactiplantibacillus plantarum,&#160;a gram-positive lactic acid bacterium13. L. plantarum is a nomadic, probiotic bacterium that resides in a wide range of environments, including the guts of humans and other vertebrates, as well as many types of food such as meat, cereals, vegetables, and fermented foods and drinks14,15,16,17. Its genome encodes a flexible, heterofermentative metabolism, allowing successful adaptation across these diverse environments. It is well-studied, widely used in the food and health industries, and generally recognized as safe by the Food and Drug Administration18,19. As such, L. plantarum has the potential to serve as a useful platform for EET-based technologies.
Recent research in L. plantarum has identified a multi-gene operon encoding a complex EET pathway originally characterized in Listeria monocytogenes13,20. In L. plantarum, the proteins synthesized from this operon facilitate EET in a bioelectrochemical system (BES) when provided the quinone 1,4-dihydroxy-2-naphthoic acid (DHNA) as an electron mediator13. The first essential protein in this pathway is a membrane-bound NADH-quinone oxidoreductase (Ndh2), which oxidizes NADH and reduces DHNA. DHNA either delivers electrons directly to an electrode or indirectly via the accessory protein PplA (Figure 1)13,21,22. Recent research indicates L. plantarum may also use other quinones that are structurally similar to DHNA as electron mediators; however, L. plantarum is incapable of producing DHNA or these alternative quinones, thus mediators must be exogenously present in the environment for EET to occur13,22,23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67204/67204fig01.jpg" /> 
Figure 1: Electron flow in Lactiplantibacillus plantarum EET. Ndh2 passes electrons from NADH to the quinone DHNA. Electrons are shuttled to the electrode to produce current, either directly by reduced quinone or indirectly through the accessory protein PplA. Abbreviations: FAD = Flavin adenine dinucleotide; FMN = Flavin mononucleotide; EET = extracellular electron transfer; NADH = reduced nicotinamide adenine dinucleotide; Ndh2 = NADH-quinone oxidoreductase; DHNA = 1,4-dihydroxy-2-naphthoic acid; PplA = phospholipase A. <a href="https://www.jove.com/files/ftp_upload/67204/67204fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this article, we provide a comprehensive protocol for using a BES-based method to characterize DHNA-mediated EET in L. plantarum. A three-electrode, two-chamber system confines bacteria to the working electrode, allowing precise control of the potential applied to the bacteria while preventing crosstalk between the working and counter electrode. We present a comprehensive protocol spanning 5 days, which covers pre experiment preparation, BES assembly, EET analysis utilizing chronoamperometry (CA) and cyclic voltammetry (CV), and post experiment sample analysis. This protocol can be applied to unravel the mechanisms of EET pathways and to build systems for electrofermentation and electrocatalysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-Dihydroxy-2-naphthoic acid (DHNA)</td>
			<td>Sigma-Aldrich</td>
			<td>281255-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 mm diameter titanium wire</td>
			<td>Thermo Fisher Scientific</td>
			<td>045485.BY</td>
			<td>Cut to size for working and counter electrodes</td>
		</tr>
		<tr>
			<td>120-C Aluminum Oxide Sheets 9&#34; x 11&#34;</td>
			<td>Johnson Abrasives</td>
			<td>10108-15</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL plastic syringes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14955457</td>
			<td></td>
		</tr>
		<tr>
			<td>3M KCl solution saturated with silver chloride</td>
			<td>Millipore Sigma</td>
			<td>60137-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>6.35-mm-thick carbon felt</td>
			<td>Thermo Fisher Scientific</td>
			<td>043200.RF</td>
			<td>Cut into 16 cm2 rounds</td>
		</tr>
		<tr>
			<td>Ag/AgCl reference electrode</td>
			<td>CH Instruments</td>
			<td>CH111</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-Tite Premium Hypodermic Needles</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-817-102</td>
			<td></td>
		</tr>
		<tr>
			<td>AlK(SO)4 * 12H2O</td>
			<td>Sigma-Aldrich</td>
			<td>237086-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium citrate tribasic</td>
			<td>Millipore Sigma</td>
			<td>A1332</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B99517</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Precision Glide Needle, 18 G x 1 inch</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Precision Glide Needle, 21 G x 2 inch</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-821-13N</td>
			<td></td>
		</tr>
		<tr>
			<td>Bel-Art SP Scienceware Cleanware Aqua-Clear Water Condtioner</td>
			<td>Thermo Fisher Scientific</td>
			<td>23-278339</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Millipore Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Millipore Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium D-(+)-pantothenate</td>
			<td>Millipore Sigma</td>
			<td>1087009</td>
			<td></td>
		</tr>
		<tr>
			<td>Casamino acids</td>
			<td>Millipore Sigma</td>
			<td>2240-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>cation exchange membrane</td>
			<td>Membranes International</td>
			<td>CMI-7000</td>
			<td>Cut into rounds fit to the BES O-ring</td>
		</tr>
		<tr>
			<td>CoCl2 * 6H2O</td>
			<td>Millipore Sigma</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4 * 5H2O</td>
			<td>Millipore Sigma</td>
			<td>C8027</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine-HCl * H2O</td>
			<td>Millipore Sigma</td>
			<td>30129</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Millipore Sigma</td>
			<td>5439001000</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-11+ Spectrophotometer</td>
			<td>Denovix</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>EC-Lab Software</td>
			<td>BioLogic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECO E 4 S heating circulator</td>
			<td>Lauda-Brinkmann</td>
			<td>Cat. No. 115 V; 60 Hz : L001191</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4 * 7H2O</td>
			<td>Millipore Sigma</td>
			<td>215422</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Millipore Sigma</td>
			<td>F8758</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>Millipore Sigma</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes with BD Micro-Fine IV Needle</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-829-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactiplantibacillus plantarum NCIMB8826</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Reference: Tejedor-Sanz et al., 2022</td>
		</tr>
		<tr>
			<td>Lactobacillus MRS Broth</td>
			<td>HiMedia</td>
			<td>M369</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Broth</td>
			<td>Milliport Sigma</td>
			<td>63011</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate anhydrous</td>
			<td>Millipore Sigma</td>
			<td>208094</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese sulfate monohydrate</td>
			<td>Millipore Sigma</td>
			<td>221287</td>
			<td></td>
		</tr>
		<tr>
			<td>mannitol</td>
			<td>Millipore Sigma</td>
			<td>M1902-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Mettler Toledo FiveEasy Benchtop pH Meter</td>
			<td>Hogentogler</td>
			<td>F20-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 * 6H2O</td>
			<td>Millipore Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 * 7H2O</td>
			<td>Millipore Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex - GV 0.22 &#181;m PVDF Membrane Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>SLGV004SL</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2 * 4H2O</td>
			<td>Millipore Sigma</td>
			<td>203734</td>
			<td></td>
		</tr>
		<tr>
			<td>MnSO4 * H2O</td>
			<td>Millipore Sigma</td>
			<td>221287</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Millipore Sigma</td>
			<td>M1442</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 gas</td>
			<td>Airgas</td>
			<td>NI UHP300</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Na2MoO4 * 2H2O</td>
			<td>Millipore Sigma</td>
			<td>331058</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Millipore Sigma</td>
			<td>238597</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Millipore Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinic acid</td>
			<td>Millipore Sigma</td>
			<td>N-0761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrilotriacetic acid (NTA)</td>
			<td>Millipore Sigma</td>
			<td>72560</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Aminobenzoic acid</td>
			<td>Millipore Sigma</td>
			<td>P9879</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP399-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Millipore Sigma</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>potentiostat</td>
			<td>BioLogic</td>
			<td>VMP-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease peptone #3</td>
			<td>Bacto</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine HCl</td>
			<td>Millipore Sigma</td>
			<td>P6280</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Millipore Sigma</td>
			<td>555682</td>
			<td></td>
		</tr>
		<tr>
			<td>RO10 magnetic stir bar platform</td>
			<td>IKA</td>
			<td>3691000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Millipore Sigma</td>
			<td>935700</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bar, egg-shaped</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-512-121</td>
			<td>Place in anodic chamber of BES</td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Millipore Sigma</td>
			<td>V-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioctic acid (&#945;-Lipoic acid)</td>
			<td>Millipore Sigma</td>
			<td>T-1395</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Millipore Sigma</td>
			<td>9136</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Millipore Sigma</td>
			<td>P4780</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Millipore Sigma</td>
			<td>V6629</td>
			<td></td>
		</tr>
		<tr>
			<td>Jacketed MCF set, 100 ml, NW25, 2 x GL14 port</td>
			<td>Adams &#38; Chittenden Scientific Glass</td>
			<td>NA</td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Millipore Sigma</td>
			<td>Y1625</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4 * 7H2O</td>
			<td>Millipore Sigma</td>
			<td>Z0251</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing mediated extracellular electron transfer in lactic acid bacteria with a three-electrode, two-chamber bioelectrochemical system

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

Chronoamperometry analysis 
The EET of L. plantarum can be observed through the chronoamperometry (CA) data depicted in Figure 3, in which the current density trace visualizes electron transfer from L. plantarum to the working electrode. We monitored the current density (j) versus time while maintaining a constant potential of +200 mV versus Ag/AgCl for 24 h. Upon injecting 20 &#181;g/mL DHNA into the stirring electrolyte solution, an abiotic DHNA oxidation spike was observed, followed by a rapid increase in biotic current density peaking at 132.0 &#177; 2.47 &#181;A/cm2 at approximately the 8 h time point. Conversely, the injection of DMSO resulted in negligible current density. These results emphasize the significance of DHNA as a necessary and efficient mediator to facilitate electron transfer between L. plantarum and the electrode. Users can adjust the current output by adjusting the concentration of DHNA in the BES. Prior research also indicates L. plantarum responds to DHNA in a dose-dependent manner across a wide range of DHNA concentrations, producing significant current in the presence of DHNA concentrations as low as 0.01 &#181;g/mL13,22.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67204/67204fig03v5.jpg" /> 
Figure 3: Chronoamperometry analysis of Lactiplantibacillus plantarum EET mediated by DHNA. DHNA (20 &#181;g/mL) or DMSO was injected into mCDM electrolytes (pH~ 6.5) with injection time identified as t = 0. j represents current density as a function of the working electrode area. Experiments were conducted at 200 mV versus Ag/AgCl with a carbon felt electrode (16 cm2)&#160;and stirring. Values are plotted as&#160;mean &#177; sd obtained in triplicate BES reactors. Abbreviations: EET = extracellular electron transfer; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide; mCDM = Chemically Defined Medium with mannitol. <a href="https://www.jove.com/files/ftp_upload/67204/67204fig03largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cyclic voltammetry analysis 
To further evaluate DHNA-mediated EET in L. plantarum, we conducted cyclic voltammetry 24 h after DHNA injection. Here we show CV traces for three conditions: L. plantarum with 20 &#181;g/mL DHNA, L. plantarum with DMSO, and media with 20 &#181;g/mL DHNA. As shown in Figure 4A, the presence of 20 &#181;g/mL DHNA in reactors containing L. plantarum resulted in a distinct increase in oxidative current at 50 mV that did not occur in the presence of DMSO alone. These data confirm that adding the redox mediator DHNA is necessary to facilitate electron transfer between L. plantarum and the anode. While we observed various smaller redox peaks in the L. plantarum + DMSO trace, these peaks were similar to the media control trace and are likely attributed to redox-active components in mCDM (Supplemental Figure S1). In Figure 4B, we compared traces of DHNA under biotic conditions (L. plantarum + DHNA) versus DHNA under abiotic conditions (Media + DHNA). While both traces exhibited a distinct DHNA oxidative peak around 50 mV, we observed a sustained increase in current beyond 50 mV only under biotic conditions. The catalytic peak reached a current density of 129 &#181;A/cm2 at 300 mV, representing a 256% increase compared to the abiotic trace. This turnover CV profile is characteristic of microbial EET27, indicating re-reduction of DHNA by L. plantarum cells in the presence of an electron source (mannitol) after oxidation of DHNA at the anode. Additionally, the abiotic trace exhibited new oxidative peaks around -240 mV and -180 mV. Prior research indicates the appearance of these peaks may be due to the degradation of DHNA into ACNQ (2-amino-3-carboxy-1,4-naphthoquinone)21,28. We did not observe these peaks in the biotic trace, indicating that the interaction of L. plantarum cells with DHNA may stabilize DHNA and prevent degradation. A point to note is that the 24 h trace for media with 20 &#181;g/mL DHNA was conducted separately according to this protocol without adding cells.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67204/67204fig04.jpg" /> 
Figure 4: Representative cyclic voltammetry traces. All CV experiments were performed in mCDM using carbon felt (16 cm2) as the working electrode at a scan rate of 2 mV/s while stirring the solution. (A) CV traces for Lactiplantibacillus plantarum with either DHNA (20 &#181;g/mL) or DMSO at t = 24 h. (B) CV traces of 20 &#181;g/mL DHNA in either L. plantarum (biotic conditions) or mCDM only (abiotic conditions) at t = 24 h. Abbreviations: CV = cyclic voltammetry; mCDM = Chemically Defined Medium with mannitol; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. <a href="https://www.jove.com/files/ftp_upload/67204/67204fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
pH analysis 
EET activity in L. plantarum resulted in a notable drop in pH over 24 h. As shown in Figure 5, the average sample pH of L. plantarum exposed to DHNA dropped to 3.33&#160;&#177; 0.01 (p = 6.85 &#215; 10-6, n = 3), while the average sample pH of L. plantarum exposed to DMSO dropped to 6.50&#160;&#177; 0.06 (p = 0.0409, n = 3). As exhibited in prior research, this drop is attributed to an increase in fermentative metabolism that occurs when L. plantarum performs EET13. L. plantarum normally metabolizes mannitol through glycolysis and fermentative pathways, which produce acetate, lactate, and ethanol as end-fermentation products and generate ATP through substrate-level phosphorylation29. Under EET conditions, metabolic flux through fermentation increases, thereby increasing the production of end-fermentation products in the BES media13. This metabolic shift causes the media pH to drop more rapidly in the reactors with DHNA compared to the DMSO control reactors.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67204/67204fig05.jpg" /> 
Figure 5: pH analysis of Lactiplantibacillus plantarum bioelectrochemical system. Samples were collected at t = 0 and t = 24 h during chronoamperometry. Values are plotted as mean &#177; sd obtained in triplicate BES reactors. Significance was determined by a one-tailed t-test. DHNA: P-value = 6.85 &#215; 10-6. DMSO: P-value = 0.0409. Abbreviations: DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. <a href="https://www.jove.com/files/ftp_upload/67204/67204fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Ingredients for preparation of mMRS media24. <a href="https://www.jove.com/files/ftp_upload/67204/67204_Table 1_ mMRS recipe.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Ingredients for preparation of mCDM media. This table is taken from Tejedor-Sanz et al.13 and Aumiller et al.25. <a href="https://www.jove.com/files/ftp_upload/67204/67204_Table 2_ mCDM recipe.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Ingredients for preparation of M9 media. <a href="https://www.jove.com/files/ftp_upload/67204/Table 3_ M9 Media.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: EC-Lab parameter settings for OCV, CA, and CV techniques. Abbreviations: OCV = open circuit voltage; CA = chronoamperometry; CV = cyclic voltammetry. <a href="https://www.jove.com/files/ftp_upload/67204/Table 4_EC-Lab Parameter Settings.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Figure S1: Representative cyclic voltammetry traces of Lactiplantibacillus plantarum with DMSO and mCDM alone. CV traces for L. plantarum with DMSO at t = 24 h and mCDM alone at t = 0 h. All CV experiments were performed using carbon felt (16 cm2) as the working electrode at a scan rate of 2 mV/s while stirring the solution. Abbreviations: CV = cyclic voltammetry; mCDM = Chemically Defined Medium with mannitol; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. <a href="https://www.jove.com/files/ftp_upload/67204/JoVE_SupplementalFig1.zip" target="_blank">Please click here to download this File.</a>

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron ...

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Research

JoVE Journal

Biochemistry

471 Views.  Rice University. Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

Video: Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Identifying G-Quadruplexes in a DNA Template Using Chemical Mapping

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Author Spotlight: Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...