Difference between revisions of "Part:BBa K1465102"

Revision as of 10:05, 20 October 2014

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.

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

Fumarate reductase activity could be demonstrated by cultivation of E. coli KRX BBa_K1465102 in our electrobiochemical H-cell reactor. We show that expression of fumarate reductase increases the electron transfer into E. coli cells via neutral red as a mediator. Results of these cultivations are shown in figure 8.

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.

@@ Line 49: / Line 49: @@
 <p>
 Upon the expression of the fumarate reductase Frd in <i>E. coli</i> we analyzed the metabolic behavior under aerobic and anaerobic conditions.
-Successful expression of the fumarate reductase Frd (<a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>) could be proven via <a href="SDSResults">SDS-PAGE</a>.
+Successful expression of the fumarate reductase Frd (<a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>) could be proven via <a href="https://parts.igem.org/Part:BBa_K1465102#SDSResults">SDS-PAGE</a>.
-Activity of the fumarate reductase displayed with HPLC analysis of fumarate consumption and succinate production in <a href="XXX">anaerobic cultivation</a> of <i>E. coli</i> was shown with <a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>. Furthermore we investigated the fumarate reductase activity in different <i>E. coli</i> strains by phenotypic MicroArray (PM) analysis with a <a href="XXX">Biolog&reg; system</a>.
+Activity of the fumarate reductase displayed with HPLC analysis of fumarate consumption and succinate production in <a href="https://parts.igem.org/Part:BBa_K1465102#AnaerobeFrdResults">anaerobic cultivation</a> of <i>E. coli</i> was shown with <a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>. Furthermore we investigated the fumarate reductase activity in different <i>E. coli</i> strains by phenotypic MicroArray (PM) analysis with a <a href="https://parts.igem.org/Part:BBa_K1465102#BiologFrdResults">Biolog&reg; system</a>. Finally we measured the electron transfer via fumarate reductase by cultivation of our self constructed <i>E. coli</i> strain in an <a href="https://parts.igem.org/Part:BBa_K1465102#H-cellResults">H-cell reactor</a>.
 </p>
@@ Line 133: / Line 133: @@
 Because the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog&reg; Microbial ID System</a> 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 <a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>. 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 <a href="https://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a> 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 <i>E. coli</i> KRX wildtype.
-    <h4>H-cell reactor</h4>
+    <h4 id="H-cellResults">H-cell reactor</h4>
      <p>
 Fumarate reductase activity could be demonstrated by cultivation of <i>E. coli</i> KRX <a href="https://parts.igem.org/Part:BBa_K1465102">BBa_K1465102</a> in our electrobiochemical <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction#text">H-cell reactor</a>. We show that expression of fumarate reductase increases the electron transfer into <i>E. coli</i> cells via neutral red as a mediator. Results of these cultivations are shown in figure 8.