Part:BBa_K3724009
Tetraheme cytochrome c protein/ quinol dehydrogenase cymA
Usage and Biology
Shewanella oneidensis MR-1 are gram-negative bacteria at the center of studies of microbial reduction due to their ability to transfer electrons extracellularly to reduce materials such as graphene oxide (GO)[1]. Such characteristics have made S. oneidensis MR-1 an organism of interest in microbial fuel cells for bioelectricity generation and potential applications in bioremediation[2]. Two extracellular electron transfer pathways have been identified in the reduction of GO by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material[3]. The DET pathway involves c-type cytochromes within the bacterial membrane including the tetraheme cytochrome c protein cymA. cymA is an inner membrane protein that is essential for the reduction of extracellular materials. It accepts electrons from the cytoplasmic menaquinone pool where these electrons are then shuttled to decaheme c-type cytochromes in the outer membrane or to electron shuttles in the periplasm for extracellular reduction[4]. Studies of the role of cymA in reduction have shown that cymA has many different terminal reductases making it a key component in the electron transfer pathway for the reduction of many different extracellular materials[2]. However, there has been some contention about whether cymA is critical for the reduction of GO or not. Recent studies have shown that cymA mutant strains of S. oneidensis have no GO reduction ability deeming cymA an essential protein in the microbial reduction of GO[5]. With this information, we sought to overexpress cymA in S. oneidensis MR-1 to increase the rate of reduction of GO. It was thought that the higher the density of the electron transport protein, cymA, in the inner membrane, the more electrons get shuttled to the outer membrane and to graphene oxide.
We therefore, synthesized the cymA gene, optimized for S. oneidensis MR-1 , and inserted it into the kanamycin resistant vector pcD8 under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin)[6].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 10
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 7
Illegal BsaI.rc site found at 583
References
[1] Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Microbial Reduction of Graphene Oxide by Shewanella. Nano Research 2011, 4, 563–570.
[2] Schwalb, C.; Chapman, S. K.; Reid, G. A. The Tetraheme Cytochrome Cyma Is Required for Anaerobic Respiration with Dimethyl Sulfoxide and Nitrite in Shewanella Oneidensis. Biochemistry 2003, 42, 9491–9497.
[3]Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella Oneidensis-Reduced Graphene Oxide Biohybrid with Enhanced Biosynthesis and Transport of Flavins Enabled a Highest Bioelectricity Output in Microbial Fuel Cells. Nano Energy 2018, 50, 639–648.
[4] Kouzuma, A.; Kasai, T.; Hirose, A.; Watanabe, K. Catabolic and Regulatory Systems in Shewanella Oneidensis MR-1 Involved in Electricity Generation in Microbial Fuel Cells. Frontiers in Microbiology 2015, 6.
[5] Jiao, Y.; Qian, F.; Li, Y.; Wang, G.; Saltikov, C. W.; Gralnick, J. A. Deciphering the Electron Transport Pathway for Graphene Oxide Reduction by Shewanella Oneidensis MR-1. Journal of Bacteriology 2011, 193, 3662–3665.
[6] Dundas, C. M.; Walker, D. J. F.; Keitz, B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates. ACS Synthetic Biology 2020, 9, 2301–2315.
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