Part:BBa_K1465102

Fumarate reductase (frd) from E. coli under the control of the T7 promoter

Fumerate reductase (frd) from E. coli under the control of the T7 promoter

Usage and Biology

Fumarate reductase is part of the anaerobic fumarate respiration in E. coli. It catalyzes the reaction from fumarate into succinate using fumarate as a final electron acceptor in anaerobic fumarate respiration. The related enzyme in aerobic respiration is the succinate dehydrogenase, which catalyses the reaction from succinate to fumarate. The electrons are transferred from succinate to FAD⁺ producing fumarate and FADH₂. The succinate dehydrogenase is also a membrane enzyme and it is part of the citric acid cycle. They both belong to the respiration complex II. Naturally there is no activity of both enzymes at the same time in E. coli cells. (Iverson et al., 1999)

Figure 1: The electron flow mediated by redox active mediator
(for example neutral red) in interaction with fumarate reductase
in E. coli cell. The electrochemical reduced mediator has to cross
outer membrane via genome integrated outer membrane porines OprF.
The fumarate reductase in the inner bacterial membrane get electrons
from the mediator for reduction of fumarate into succinate
in the cytoplasm. Excretion of succinate is avoid becuase of
knocked out C4 carboxylate antiporter DcuB.

In our project we used the fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator crosses the outer membrane of E. coli through the outer membrane porine OprF (BBa_K1172507). Mediators adsorb at the inner membrane and transfer electrons to the fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. This process has been shown for the fumarate reductase in Actinobacillus succinogenes by Park et al..
Succinate can serve as a substrate for the succinate dehydrogenase, which catalyzes the oxidation of succinate into fumarate again. So we want to create a loop in the citric acid cycle between fumarate and succinate generating FADH₂ as a reductive power in the cell. Electrons are transferred to FAD+, which generate proton translocation from the cytoplasm into the periplasmatic space. The proton motoric force leads to ATP production. To conclude the mediator-dependent activity of the fumarate reductase could serve as an energy source for bacterial cells.
We compared the activity of the fumarate reductases (Frd) from Escherichia coli KRX working with different mediators, for example neutral red or bromphenol blue, as electron donors in our electrobiochemical H-cell reactor.
For optimal functionality of BBa_K1465102 E.coli KRX ΔdcuB::oprF has to be used. Combination of this strain with strong expression of the fumarate reductase ensures efficient electron transfer into bacterial cells. Construction of E.coli KRX ΔdcuB::oprF occured with special designed deletion cassette using primer BBa_K1465107, BBa_K1465108, BBa_K1465109 and BBa_K1465110.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1352
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 666
Illegal AgeI site found at 741
Illegal AgeI site found at 3023
1000
COMPATIBLE WITH RFC[1000]

Results

Upon the expression of the fumarate reductase Frd in E. coli we analyzed the metabolic behavior under aerobic and anaerobic conditions. Successful expression of the fumarate reductase Frd (BBa_1465102) could be proven via SDS-PAGE. Activity of the fumarate reductase displayed with HPLC analysis of fumarate consumption and succinate production in anaerobic cultivation of E. coli was shown with BBa_1465102. Furthermore we investigated the fumarate reductase activity in different E. coli strains by phenotypic MicroArray (PM) analysis with a Biolog® system. Finally we measured the electron transfer via fumarate reductase by cultivation of our self constructed E. coli strain in an H-cell reactor.

SDS-PAGE

The fumarate reductase could be detected in purified membrane and periplasmatic protein fractions. Proteins were fractioned by cold osmotic shock of E. coli KRX at different steps after induction of protein expression.
SDS-PAGE shows the expression of fumarate reductase in E. coli KRX under control of the T7 promotor (BBa_1465102). Fumarate reductase with a total molecular mass of 121 kD consists of four subunits. There are two large catalytic and water-soluble subunits, flavoprotein (66 kDa) and iron-sulfur protein (27 kDa). Two small membrane associated subunits (15 and 13 kDa) are not detectable in SDS-PAGE. (Iverson et al., 1999).
Figure 2 shows the result of the SDS-PAGE of isolated membrane proteins via cold osmotic shock of E. coli KRX with BBa_1465102. Membrane protein fractions at different times after induction of BBa_1465102 are displayed.

Figure 2: Results of the SDS-PAGE of purified membrane protein from Escherichia coli BBa_1465102 via cold osmotic shock. Expected size of fumarate reductase subunits A and B are 66 kDa and 27 kDA respectively. Increasing band at 66 kDa could be recognized attributable to subunit A. Increasing band between 40 and 55 kDa could be referable to dimer or trimer build-up by subunit B, C and D.

SDS_PAGE shows a significantly higher protein concentration in membrane extracts from E. coli overexpressing frd under control of T7 promoter (BBa_1465102) after induction. This is caused by usually higher membrane protein concentration of cultivated cells attributable to higher optical density. Nevertheless, a strong overexpression band can be observed at the expected Frd size (subunit A) of about 66 kDa for BBa_1465102, which can be traced back on the strong expression and overproduction of Frd. Besides an overexpression band can be recognized at a size of about 45 kDA. This could be explained by dimer formation of two or three Frd subunits, for example subunit B (27 kDa) with subunit C oder D (13 and 15 kDA). That implies that subunit B build up stable dimers with small membrane associated subunits C and D of about 40 kDa respectively 42 kDa. There seems to be no dimers of subunit A with other parts of the Frd.

Anaerobic cultivation

We cultivated E. coli KRX with BBa_1465102 under anaerobic conditions to characterize the activity of the fumarate reductase Frd (BBa_1465102) under control of the T7 promotor. We used M9 minimal medium with 50 mM xylose as carbon source and 50 mM fumarate as substrate of the fumarate reductase Frd. We focused on growth characteristic and metabolite analysis. Under anaerobic conditions E. coli cells use different alternative electron acceptors instead of oxygen. In part the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential pathways for bacteria to release their electrons, for example anorganic compounds like nitrate (NO₃^-) or sulfate (SO₄^2-). As a result of this the expected level of produced succinate is very low. We analyzed fumarate and succinate concentrations in the culture supernatant via HPLC analysis.

We expect mid-level succinate production of the E. coli KRX wildtype under anaerobic conditions. Strong expression of Frd (BBa_1465102) leads to an increasing succinate production and fumarate consumption.

Figure 3: Results of the anaerobic cultivation of Escherichia coli KRX wildtype in M9 minimal medium with 50 mM xylose and 50 mM fumarate. Induction occurred after 72 h with 0.1% rhamnose and 500 μM IPTG. Fumarate and succinate concentration were measured duplicated via HPLC.

Figure 4: Results of anaerobic cultivation of Escherichia coli KRX wildtype with strong expression of frd (BBa_1465102) in M9 minimal medium with 50 mM xylose and 50 mM fumarate. Induction occurred after 72 h with 0.1% rhamnose and 500 μM IPTG. Fumarate and succinate concentration were measured duplicated via HPLC.

Figure 5: Growth of the Escherichia coli KRX wildtype with strong expression of frd (BBa_1465102) compared to Escherichia coli KRX wildtype under anaerobic condition in M9 minimal medium with 50 mM xylose and 50 mM fumarate. Induction occurred after 72 h with 0.1% rhamnose and 500 μM IPTG. Fumarate and succinate concentration were measured duplicated via HPLC.

Figure 6: Comparison of fumarate consumption and succinate production in anaerobic cultivation of the Escherichia coli KRX wildtype and i>Escherichia coli KRX wildtype with strong expression of frd (BBa_1465102) in M9 minimal medium with 50 mM xylose and 50 mM fumarate. Induction occurred after 72 h with 0.1% rhamnose and 500 μM IPTG. Fumarate and succinate concentration were measured duplicated via HPLC.

As expected figure 3 shows the production of succinate by the Escherichia coli KRX wildtype under anaerobic condition with 50 mM xylose and 50 mM fumarate as sole carbon sources. Under anaerobic conditions fumarate respiration takes place. Growth displayed via OD₆₀₀ shows rise in the first three days of anaerobic cultivation. After that cells were induced via 0.1% rhamnose and 500 μM IPTG. This leads to a reduction in growth because of the expression load and the toxic effect of IPTG. Cultivation of the E. coli wildtype also should be induced because of better comparability. The same effect could be observed in cultivation of E. coli with BBa_1465102, as shown in figure 4. There is also a drop in growth detectable. Comparison of both growth characteristics are shown in figure 5. It could be proven that there are no distinct differences in growth betweenE. coli KRX wildtype and E. coli KRX with BBa_1465102. So statement about fumarate reductase activity in E. coli KRX with BBa_1465102 as measured by succinate production and fumarate consumption is possible.
Figure 6 shows comparison of succinate production and fumarate consumption between E. coli KRX wildtype and E. coli KRX with BBa_1465102. This illustrates an increasing succinate production by E. coli KRX with BBa_1465102 after induction of T7 promotor. A higher value of succinate could be achieved after 8 days of anaerobic cultivation and also lower fumarate level was shown compared to E. coli KRX wildtype.

Phenotypic characterization with Biolog® system

Biolog® Microbial ID System is a simple and fast method for the characterization of different bacteria, yeast and fungi species. It is based on respiration in the presence of different metabolic relevant substances for example carbon sources, nitrogen sources or toxines. The system uses redox chemistry wherein a tetrazolium dye is reduced when significant respiration occurs. Tetrazolium dye change its colour, because the cells generate reductive conditions in the course of electron transport chain. Tetrazolium cation is reduced to formazan by dehydrogenase from respiratory complex I. This allow a statement if respiration occurs in the presence of different substances.

We tested the influence of fumarate on the Escherichia coli KRX wildtype and E. coli KRX with BBa_1465102. To show activity of fumarate reductase under controll of the T7 promotor (BBa_1465102), we incubated cells with fumarate in a Biolog® system for 48h. Results of the Biolog ® analysis are shown in figure 7.

E. coli KRX wildtype and E. coli KRX with BBa_1465102 show respiratory activity in the presence of fumarate. As unexpected E. coli KRX wildtype shows higher activity on fumarate as compared to BBa_1465102. Respiration of E. coli KRX with BBa_1465102 seems to be fewer and occurs subsequently in comparison to wildtype.

Figure 7: Results of Biolog® analysis of E.coli KRX with BBa_1465102 in comparison to Escherichia coli KRX wildtype. Respiratory activity is shown under influence of fumarate.

Figure 7 shows the respiratory activity of E. coli KRX with strong expressed BBa_1465102 in comparison to the E. coli KRX wildtype. As expected the wildtype shows higher activity in presence of fumarate as the fumarate reductase expressing strain. This effect is attributed to the fumarate reductase activity.
Because the Biolog® Microbial ID System is based on the activity in the electron transport chain and reduction of tetrazolium dye, direct conclusions about fumarate consumption are not possible. But there are indirect hints for fumarate reductase activity via the strong expression of BBa_1465102. Fumarate reductase naturally gets electrons out of electron transport chain and transfer them to fumarate generating succinate, which is transported out of the cells under anaerobic conditions. Under aerobic conditions succinate dehydrogenase oxidize succinate to fumarate again. This created loop by expression of BBa_1465102 leads to lower electron transfer activity in the respiratory chain. So there are less electrons available for reduction of the extracellular tetrazolium dye compared to the E. coli KRX wildtype.

H-cell reactor

There is evidence for activity of the Fumarate reductase which could be demonstrated by cultivation of E. coli KRX BBa_K1465102 in our electrobiochemical H-cell reactor. We showed that expression of fumarate reductase has an impact on the NAD/NADH levels in the E. coli cells that were cultivated with the mediator neutral red. The results of these cultivations are shown in figures 8 and 9.

Figure 8: Cultivation of the E. coli KRX ΔdcuB::oprF strain in M9 minimal media with xylose (50 mM) and 100 µM neutral red added. During the cultivation there was set a potential of -400 mV on the H-cell achieved by the chronoamperometric method. The figure shows the optical density, xylose concentration, and the NAD/NADH level during the cultivation, plotted against time.

Figure 9: Cultivation of the E. coli KRX WT in M9 minimal medium with xylose (50mM). During the cultivation there was set a potential of -400 mV on the H-cell achieved by the chronoamperometric method. The figure shows the optical density, xylose concentration, and the NAD/NADH level during the cultivation, plotted against time.

The NAD/NADH level was measured with the GloAssay™ by Promega. There are only slightly differences in the levels of NAD and NADH in both cultivations. The growth curve in figure 8 shows a faster increase in comparison to the growth curve in figure 9. This indicates that the growth might be enhanced by neutral red and the applied current of -400mV.

Methods

SDS-PAGE

Pouring the polyacrylamide gel:

For each separating gel (12 %) aliquote:

1.35 ml Bisacrylamid/Acrylamid (0.8 % , 30 %, at the ratio of 37.5:1)
0.675 ml H₂O
0.675 ml 1.88 M Tris-HCl (pH 8.8)
0.675 ml 0.5 % SDS

Add 120 µl 10 % ammonium persulfate and 37,5 µl TEMED to each aliquote and mix
Pour the solution quickly into the gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel
Layer isopropanol on top of the gel
Leave the separating gel at room temperature for >60 minutes to polymerize

Remove isopropanol and wash the surface of the separating gel with H₂O. Wait until the surface is dry
For each stacking gel (5 %) aliquote:

0.309 ml Bisacrylamid/Acrylamid (0.8 % , 30 %, at the ratio of 37.5:1)
0.726 ml H₂O
0.375 ml 0.625 M Tris-HCl (pH 6.8)
0.375 ml 0.5 % SDS

Add 60 µl 10 % ammonium persulfate and 22,5 µl TEMED to each aliquote and mix
Insert comb without getting bubbles stuck underneath
Leave the gel at room temperature for >60 minutes to polymerize
For storage:

Remove sealing and store the gel wrapped in moistened paper towel at 4 °C

Preparing the sample:

Mix your protein mixture 3:1 with PBJR buffer (15 µl protein solution + 5 µl PBJR buffer)
Heat for 5 minutes at 95 °C

Running the gel:

Remove sealing, put the polymerized gel into gel box and pour SDS-PAGE running buffer into the negative and positive electrode chamber
Remove comp without destroying the gel pockets
Pipet the SDS running buffer in the gel pockets up and down for flushing the gel pockets
Pipet slowly 20 µl of the sample into the gel pockets
Make sure to include at least one lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of the sample
Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel
Raise amperage up to 20 mA for running the separating gel
When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply

Staining the polyacrylamide gel (Colloidal Coomassie Brilliant Blue staining):

After finishing the SDS-PAGE remove gel from gel casting form and transfer it into a box
Add 100 ml of the Colloidal Coomaassie Brilliant Blue staining solution to your polyacrylamid gel
Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation
Remove the staining solution
Wash the gel with 7 % (v/v) acetic acid in H₂O for decoloration
Incubate the gel in H₂O (2-6 h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one

Cold osmotic shock

Release of periplasmic protein fraction from E. coli by cold osmotic shock
Modified protocol from Neu & Heppel, 1965:

Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
Discard the entire supernatant
Resuspend the cells in ice-cold Cell Fractioning Buffer 1. The resulting volume should be 1/4 of the former suspension volume
Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
Discard the entire supernatant
Resuspend the cells in ice-cold Cell Fractioning Buffer 2. The resulting volume should be 1/4 of the former suspension volume
Incubate for 10 up to 20 min on ice under regular invertion
Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
Save the supernatant, which contains the periplasmatic proteins and membrane proteins

Anaerobic cultivation

Whole anaerobic work takes place in a two-hand Glove Bag.
All media and buffer has to be degased with nitrogen (N₂) gas via a sparger befor starting cultivation.
Grow preculture under aerobic condition at 37°C.
Cells were cultivated in a gas-tight 15 ml tube additional sealed with parafilm.
Cultivation volume is about 10 mL. Fill cultivation tube with 8.5 ml steril degased cultivation medium.
When preculture reaches OD₆₀₀ of 0.6-0.8, take 1.5 ml into a steril tube.
Centrifugate 1 min at 5,000 rpm and discard complete supernatant.
resuspend pellet in 1.5 ml steril degased PBS buffer.
Repeat previous step but resuspend in steril, degased cultivation medium. This two steps avoid residual oxygen in the inoculum.
Inoculate culture with 1.5 ml washed and resuspended cells at OD₆₀₀ of 0.1.
Tubes are filled and opened for sampling only under nitrogen atmosphere.
Tubes are incubated in a shaker at 37°C.
At the beginning of anaerobic cultivation residual oxygen from preculture would be consumed. Therefore faster growth could be observed in the first period of cultivation until cells shift to anaerobic metabolism.
There should be more frequent sampling at the beginning about 4 to 6-hour intervalls (two times) after inoculation.
Regular sampling takes place at 1-day intervalls.
Sampling volume is about 1 mL, using 500 μl for OD₆₀₀ measurement and 500 μl for HPLC analysis.
After sampling process tubes are overflowed with nitrogen gas, tightly closed and sealed with parafilm again.
Tubes are further incubated in a shaker at 37°C.

Biolog System

This protocol is based on the technical guide from Biolog

Grow the strain of interest on a BUG+B (Biolog Universal Growth Medium + 5 % Sheep blood) agar plate by streaking out for single colonies
Incubate overnight at 37 °C
Pick single colonies with sterile swab
Transfer them into sterile tube containing 10 ml 1.0x IF-0a (mix 8 ml 1.2x IF-0a with 2 ml sterile water) to adjust transmittance to 42 %. This creates solution A.
Mix 144 μl Dye Mix A for Gram-negative bacteria and 1.856 ml sterile water with 8 ml 1.2x IF-0a solution. This creates solution B.
Mix 1.8 ml of solution A with 9 ml of solution B to achieve a final cell density of 85 % transmittance. This creates solution C.
Inoculate Biolog PM plate (96 wells) with 100 μl suspension C per well incubate loaded plate for 48 h in the OmniLog PM system

Glo™Assay by Promega

Basic principle of the enzymatically detection assay:

NAD/NADH-Glo™ Assay

⁺

Protocol:

1 ml of bacteria culture (OD₆₀₀= 0,375 ≈ 3 • 10³ cells)get pelleted by centrifugation
Remove supernatant and add 300 µl bicarbonatebuffer + 1% DTAB (dodecyl(trimethyl)azanium bromide)
Mix thoroughly for cell lysis
Assay the neutralized samples using the NAD/NADH-Glo Assay by transferring 30 µl of each sample to the wells of a 96-well white luminometer plate and add 30µl of NAD/NADH-Glo Detection Reagent.
Incubate at room temperature and read luminescence after 30 to 60 minutes.
Determine NAD/NADH ratios by comparing RLU, or calculate the concentrations by comparison to a standard curve.
This method is used, if you want to measure the total amount of NAD⁺ and NADH. For the determination of the individual NAD₊ and NADH concentrations follow the description below.

Individual determination of NAD⁺ and NADH

After the cells got mixed briefly transfer two 100µl aliquots to each of two new tubes.(R1 and R2)
Add 100µl HCl (0.4M) to R1 for acid treatment
Heat both aliquots at 60 °C for 15 minutes
Cool at room temperature for 10 minutes
Neutralize R1 with 100 µl trizma base (0.5M). This sample now contains the oxidized form NAD.
Neutralize R2 with 200µl of a 1:1 mixture of HCl (0.5M) and trizma base (0.5M). This sample now contains the oreduced form NADH
Both samples can be assayed as already described.

GloMax® Discover Multimode-Reader

References

Iverson et al., 1999. Structure of the Escherichia coli Fumarate Reductase Respiratory Complex. Science, vol. 284, pp. 1961-1966
Janausch, 2001. Rekonstitution des Fumaratsensors DcuS in Liposomen und Transport von Fumarat und Succinat in Escherichia coli. Doctoral dissertation at Johannes Gutenberg-Universität Mainz, Germany
Gottschalk et al., 1986. Bacterial Metabolism. Springer-Verlag, New York, Berlin, Heidelberg, Tokyo
Park et al., 1999. Utilization of Electrically Reduced Neutral Red byActinobacillus succinogenes: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation. Journal of Bacteriology, vol. 181, pp. 2403-2410
Richardson et al., 1999. Bacterial respiration: a flexible process for a changing environment. Microbiology, vol. 146, pp. 551-571
Unden et al., 1997. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochimica et Biophysica Acta, vol. 1320, pp. 217-234
Stycharz, S. M., Glaven, R. H., Coppi, M. V., Gannon, S. M., Perpetua, L. A., Liu, A., Nevin, K. P. &amp. Lovley, D. R. (2011):Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens. In: Bioelectrochemistry 80, pp. 142 - 150.
Dantas, J. M., Tomaz, D. M., Morgado, L. & Salgueiro, C. A.(2013): Functional charcterization of PccH, a key cytochrome for electron transfer from electrodes to the bacteium Geobacter sulfurreducens. In: FEBS Letters 587, pp. 2662 - 2668.
Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., Weigele, P., Groves, J. T., Ajo-Franklin, C. M. (2010): Engineering of a synthetic electron conduit in living cells. In: PNAS 107, pp. 19213–19218.