Cytochromes

Overview

Figure 1: Extracellular electron transfer via cytochromes in E. coli with a minimal set of proteins from Shewanella oneidensins MR-1.

To enable transfer of electrons from the general metabolism to the outside of the cell the mtrCAB operon from [http://www.ncbi.nlm.nih.gov/genome/1082?project_id=57949 Shewanella oneidensis MR-1] was heterologously expressed in E. coli. This operon encodes for a minimal set of genes required to build an electron shuttle pathway via different c-type cytochromes. Electrons from native E. coli protein [http://www.ncbi.nlm.nih.gov/protein/EGT66377.1 NapC] are passed to periplasmic [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA], which transports them to the outer membrane protein [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB]. Via the membrane-bound [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] the corresponding electrons can be transfered to extracellular electrone acceptors like the anode of a microbial fuel cell.
The mtrCAB cluster contains two illegal restriction sites, which where removed by generating a silent mutation via overlap-extension-PCR. The resulting three fragments were combined and ligated with [http://parts.igem.org/Part:pSB1C3 pSB1C3] by Gibson assembly. Subsequently this gene cluster was combined with three different promotors and ribosome binding sites of varying strength. The functional expressin of the cytochromes could not be experimentaly verified. Furthermore it was attempted to clone the ccmAH cluster as well, which was unsuccesfull. This is, however, a minor issue, since the microbial fuel cell will work under anareobic conditions. For a correct heme insertion into the decaheme cytochromes [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] and [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] the cytochrome c maturation machinery is required. The corresponding genes are naturally expressed in E. coli under anaerobic conditions, for aerobic expression they have to be expressed via plasmid.

Theory

Cell membranes work as a natural insulator and prevent the flow from electrons out of the cell. To enable transfer of electrons from the general metabolism to the outside of the cell we had to alter the membrane of our organism E. coli without disturbing cell growth, stability and metabolism.
Some species from the genera Shewanella and Geobacter have developed different mechanisms to allow extracellular electron transfer. Members of the Shewanella species are the gram-negative γ-proteobacteria and are known for their respiratory versatility. They are reported to be using over 20 terminal electron acceptors for respiration (Nealson, Scott, 2003). Shewanella oneidensis MR-1 expresses many c-type cytochromes, membrane-bound redox-active proteins or soluble periplasmatic proteins, which play an important in the electron transport in the bacterial respiration and photosynthesis.
There have been already numerous approaches, where the complex MtrCAB was introduced into E.coli strains and the genes could be successfully expressed in E. coli under anaerobic cultivation (Goldbeck et al. 2012)

Genetic Approach

Isolation of mtrCAB

Figure2: The genomic region of the mtrCAB operon is shown with a screenshot from [http://ncbi.nlm.nih.gov NCBI].

Figure3: Overview on the illegal restriction sites within the mtrCAB operon and the resulting fragments from the overlap extension PCR approach. The colored overlaps indicate homologous sequences,that facilitate the religation via Gibson assembly. The parts labeled 'pSB1C3 prefix' and 'pSB1C3 suffix' are overlaps homologous to the vectors prefix and suffix, respectivly.

As shown in Figure2 the three corresponding genes are organized in an operon in the donor organism Shewanella oneidensis MR-1 and could therefore amplified altogether. Since two illegal PstI restriction sites are present in this cluster it had to be subdivided into three fragments. The illegal restriction sites were subsequently deleted by incorporating a silent mutation via overlap extension PCR. Eventually the fragments should be joined back together and ligated with the shipping backbone <bbpart>pSB1C3</bbpart> via Gibson assembly.
This approach required specifically designed primers that can bind the targeted genetic sequence and hold an overlap to facilitate ligation with the other fragments and the backbone.Hence, eight primers were designed, two for each fragment of the mtrCAB operon and two for the <bbpart>pSB1C3</bbpart> vector to generated proper backbone-overlaps as well. The resulting fragments are visualized in Figure 3. The colored overlaps indicate homologous sequences,that facilitate the religation via Gibson assembly. The parts labeled 'pSB1C3 prefix' and 'pSB1C3 suffix' are overlaps homologous to the vectors prefix and suffix, respectivly. The genes and resulting fragments are listed in the table below. The fragment sizes incorporate the aforementioned primeroverlaps of 20 bp each.

Promoter strength

Due to the complex interaction between the Mtr proteins among themselves and with the Ccm system the transcriptional balance is crucial. Previous works proved that only a small window for optimal MtrCAB expression exists. Most important, the synthesis of the mature holocytochromes MtrC and MtrA actually decreases at a high corresponding promoter activity. A possible explanation for this is the low processivity of the Ccm machinery, which cannot cope with a high level of apocytochromes resulting in incompletely matured apocytochromes, which are then degraded by periplasmatic proteases. Furthermore a too low level of Ccm proteins inhibits the maturation of the apocytochromes, whereas expression above a certain treshold decreases the overall efficency of the Ccm system.
Since the genes are expressed heterologously via plasmid promoter strength plays a key role in a controlled and therefore succesfull expression. To cover a wide range of expression levels three different promoters, with varying strength and ribosome binding sites where combined with the genes. An overview is given in the table below.

Protein Overview

Figure 4: Extracellular electron transfer via cytochromes in E. coli with a minimal set of genes from Shewanella oneidensins MR-1.

Shewanella oneidensis MR-1 expresses many c-type cytochromes, membrane-bound redox-active proteins or soluble periplasmatic proteins, which play an important in the electron transport in the bacterial respiration and photosynthesis. One of the electron-transfer models is based on c-type cytochromes encoded by the mtrCAB gene cluster (Myers, Myers, 1997). Although additional proteins like OmcA and NapC are involved in the electrone transport chain, a minimal set of the three proteins MtrC, MtrA and MtrB is sufficient to heterologously express this pathway in E. coli (Jensen et al. 2010).
The process of the electron transport is illustrated in Figure 4. NapC ist naturally expressed from E.coli and interacts very well with the proteins from S. oneidensis. The inner membrane cytochrome c NapC accepts electrons generated by the reduction of menaquinone to menaquinol. The electrons are then passed from CymA to the periplasmic decaheme cytochrome c [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] in the periplasm, which transports them to [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB], a 72-kD β-barrel outer membrane protein, which is physically connected with [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC], a 69-kDa membrane-bound decaheme cytochrome c. From MtrC the corresponding electrons can be transfered to extracellular electrone acceptors such as iron oxid, or, as in our case, an anode in a microbial fuel cell.
The CcmAH complex composed of the eight membrane proteins CcmABCDEFGH is essential for the biosynthesis of the proteins decaheme cytochromes MtrA and MtrC. The system first loads the heme into the periplasm and catalizes the formation of thioester bonds that link the heme to two cystein residues in the apocytochrome. Afterwards the axial ligands are located towards the heme and the holocytochrome is folded.(Sanders et al. 2010). Naturally these proteins are expressed by E. coli solely under anaerobic conditions.

Localisation

• The genes mtrA, mtrB and mtrC are organized in an operon (Beliaev et al. 1998, 2001) in the genome of Shewanella oneidensis MR-1 and could therefore expressed in one piece. However, because of two illegal PstI restriction sites at 1793bp and 2078bp, we used overlap extension pcr with primers suitable for Gibson assembly to amplify three individual fragments, where the restriction site in question was deleted by a silent mutation. The fragments were joined back together and ligated into the <bbpart>pSB1C3</bbpart> vector via Gibson assembly in one step.

Mechanism

• mtrCAB and ccmAH are expressed in the cytosol. The proteins are translocated into the periplasma via the sec secretion system.
• The Ccm proteins form a large complex and accomplish the maturation of the decaheme proteins MtrA and MtrC. The system first loads the heme into the periplasm and catalizes the formation of thioester bonds that link the heme to two cystein residues in the apocytochrome. Afterwards the axial ligands are located towards the heme and the holocytochrome is folded.(Sanders et al. 2010).
• In the following MtrB is transfered into the membrane, whereas MtrC is translocated at the outer membrane. MtrA remains in the periplasm.

Difficulties

• MtrA seems to be necessary for the stability of MtrB (Schicklberger et al. 2011)
• MtrB itself is required for the correct location and incorporation of MtrC (Meyer CR, Meyer JM, 2002) and is involved in the interaction between MtrC and MtrA (Beliaev AS, Saffarini DA1, 1998).
• Furthermore extensive postranslational processing is required for correct incorporation of all hemes, folding and localisation of the cytochromes. Considering these difficulties the expression of even a single cytochrome is a major challenge (Jensen et al. 2010).

Results

Genetics

• The isolation of the mtrCAB cluster from the genome of Shewanella and the deletion of illegal restriction sites could be accomplished
• The gene cluster was succesfully ligated into the shipping vector <bbpart>pSB1C3</bbpart> forming the biobrick <bbpart>BBa_K1172401</bbpart>
• The gene cluster was ligated into three different expression vectors forming the following devices
  • <bbpart>BBa_K1172403</bbpart>, <bbpart>BBa_K1172404</bbpart>, <bbpart>BBa_K1172405</bbpart>

Characterisation

• A of periplasmatic and membrane fractions did not yield postive results. Figure 1 shows an image of t
• To prove the expression of the Mtr proteins, colonies transformed with <bbpart>BBa_K1172401</bbpart> as a control, besides <bbpart>BBa_K1172403</bbpart> and <bbpart>BBa_K1172405</bbpart> (undinduced) were cultivated anaerobically. Membrane and periplasmatic fractions were isolated by Cold osmotic shock fractioning.
• Subsequently these fractions were analyzed by SDS-PAGE; the results are shown in Figure 1.
  • The membran fraction should contain [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB] and [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] with sizes of 72 kDa and 69 kDa, respectivly.
  • The periplasmatic fraction should only contain [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] (32 kDa).

Figure 1: Image of a SDS-PAGE with periplasmatic and membrane fractions of colonies transformed with pSB1C3::mtrCAb as a control, besides K608006::mtrCAB and K525998::mtrCAB (undinduced) for analysis. Ladder: PageRuler unstained Protein Ladder (Fermentas)

• The SDS-PAGE did not yield a positiv result. No significant difference at the relevant protein sizes could be observed

• The redox activity of [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] and [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] was probed via absorption spectroscopy and did not show significant indications for a positiv result.

Conclusion

• The electrone transfer system from Shewanella oneidensis MR-1 is known to be suitable for the usage in a microbial fuel cell.
• The expression, heme-loading, correct folding and localisation of the cytochromes is very complex and hard to achieve.

References

• Beliaev AS, Saffarini DA (1998)Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction. [http://jb.asm.org/content/180/23/6292.short J Bacteriol 180:6292–6297]

• Beliaev, A. S., D. A. Saffarini, J. L. McLaughlin, and D. Hunnicutt. 2001. MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2001.02257.x/full Mol. Microbiol. 39:722-730]

• Grove, J., Tanapongpipat, S., Thomas, G., Griffiths, L., Crooke, H., and Cole, J. (1996)Escherichia coliK-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1996.383914.x/abstract Mol. Microbiol. 19, 467−481]

• Jensen HM, Albers AE, Malley KR, Londer YY, Cohen BE, et al. (2010) Engineering of a synthetic electron conduit in living cells. [http://www.pnas.org/content/107/45/19213.short Proc Natl Acad Sci USA. 10.1073/pnas.1009645107 Proc. Natl Acad. Sci. USA 107, 19213–19218 (2010).]

• Myers CR, Myers JM (2002) MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1. [http://aem.asm.org/content/68/11/5585.short Appl Environ Microbiol68:5585–5594]

• Sanders, C., Turkarslan, S., Lee, D.-W., and Daldal, F. (2010) Cytochromecbiogenesis: the Ccm system. [http://www.sciencedirect.com/science/article/pii/S0966842X10000442 Trends Microbiol. 18, 266−274]

• Schicklberger, M., Bucking, C., Schuetz, B., Heide, H., and Gescher, J. (2011) Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the beta-barrel protein MtrB. [http://aem.asm.org/content/77/4/1520.short Appl. Environ. Microbiol. 77, 1520−1523]

• Thony-Meyer L, Fischer F, Kunzler P, Ritz D, Hennecke H (1995)Escherichia coligenes required for cytochrome c maturation. [http://jb.asm.org/content/177/15/4321.short J Bacteriol 177:4321–4326]