Cytochromes

Overview

Figure 1: Extracellular electron transfer via cytochromes in E. coli with a minimal set of genes 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. For a correct heme insertion into the decaheme cytochromes MtrA and 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. 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.

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. In Shewanella oneidensis MR-1 this is achieved via different c-type cytochromes, which shuttle the electrons along a defined molecular route from the cytoplasma and the inner membrane to the outside of the cell during anaerobic respiration. This pathway is very well understood and characterized.
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. One of the electron-transfer models points out at the c-type cytochromes encoded by the omcA-mtrCAB gene cluster (Myers, Myers, 1997). CymA is the inner membrane cytochrome c and officiates as the electron-acceptor. Analagoical role plays in E. coli NapC and further, to date unidentified, proteins. The electrons are then transferred through from CymA to the MtrA in the periplasm. From MtrA, electrons are transferred to the proteins in the outer membrane, within the MtrCAB complex, and further to extracellular electron acceptors (Goldbeck et al., 2012). MtrA is a periplasmic decaheme cytochrome c of the mass 32-kDa, MtrB is a 72-kD β-barrel outer membrane protein and MtrC is a 69-kDa membrane-bound decaheme cytochrome c. MtrC requires MtrB for the correct assembly within the outer membrane(Myers, Myers, 2002). In addition, MtrB requires MtrA for its stability. Furthermore, essential for the biosynthesis of the proteins MtrA and MtrC are the cytochrome c maturation (ccm) genes, which encode eight membrane proteins CcmABCDEFGH. 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) Previous work suggests that a working electron transfer chain can be achieved by a minimal set of three genes, the periplasmatic decaheme [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA], the outer membrane β-barrel protein [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB] and the outer membrane cytochromes [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC]. MtrA interacts with at least one native redox protein, f.e. NapC and can therefore start the transfer of electrons. (Jensen et al. 2010) Additionally another set of genes, the cytochrome c maturation genes (ccmABCDEFGH), are required for correct protein localization and heme insertion into MtrA and MtrC. Under anaerobic conditions these genes are naturally expressed in E. coli, whereas under aerobic conditions we had to co-express them. For aerobic growth the cells were transformed with both plasmids, containing the cytochrome-cluster, as well as the ccmAH-cluster . By this approach extracellular electron transfer should be possible in E.coli and allow the use of this genetically engineered strain in a microbial fuel cell. (Thony-Meyer et al. 1995)

Genetic Approach

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

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 pSB1C3 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, as well as the backbone. Hence, eight primers were designed, two for each fragment of the mtrCAB operon and two for the pSB1C3 vector to generated proper backbone-overlaps as well.

Figure3: The illegal restriction sites in the mtrCAB gene cluster are visualised by a screenshot from [http://www.scied.com/pr_cmbas.htm CloneManager].

The resulting fragments are listed in the table below.

• Fragment1
  • Start to first PstI
  • Size: 1833bp (incl. overlaps for Gibson assembly

• Fragment2
  • PstI to PstI
  • Size: 321bp (incl. overlaps for Gibson assembly)

• Fragment3
  • Second PstI to end
  • Size: 3057bp (incl. overlaps for Gibson assembly)

Genes overview

• [http://www.ncbi.nlm.nih.gov/gene/1169552 mtrC]
  • Size:2016

• [http://www.ncbi.nlm.nih.gov/gene/1169551 mtrA]
  • Size:1002

• [http://www.ncbi.nlm.nih.gov/gene/1169550 mtrB]
  • Size:2094

Protein Overview

• [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC]
  • 671 aa
  • Size:69 kD
  • outer membrane decaheme tyoe c cytochrome
  • needs Ccm-machinery for heme insertion
  • needs MtrB for correct localisation

• [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA]
  • 333 aa
  • Size:32 kD
  • periplamatic decaheme type c cytochrome
  • needs Ccm-machinery for heme insertion

• [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 mtrB]
  • 697 aa
  • Size:72 kD
  • 2 strand β-barrel membrane protein
  • needs MtrA for stability

• CcmAH
  • cytochrome c maturation cluster
  • loads heme to apocytochromes

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 pSB1C3 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 pSB1C3 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 SDS-PAGE of periplasmatic and membrane fractions did not yield postive results.
• The redox activity of MtrA and MtrC was probed via absorption spectroscopy and did not show any activity.

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]