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 the cyma gene 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].
Results
Optical density measurements (O.D.600)
Previous studies have shown that the magnitude of reduction of graphene oxide can be measured using optical density measurements at a wavelength of 600 nm. It has also been shown that the presence of graphene oxide has no significant effect on the growth rate of S. oneidensis MR-1.[7] So, we utilized this technique to measure and compare the magnitude of reduction with our different transformed strains and the wildtype.
Expression of our genes in our IPTG-inducible vector, pcD8, was initially induced with 1.5mM IPTG. Immediately following induction, reduction of graphene oxide was carried out under constant shaking at 200 rpm, and the O.D.600 was measured every hour for 48 hours for TSB only, graphene oxide only, graphene oxide and TSB only, and graphene oxide and TSB each with cymA and the wildtype. Here, the TSB only , graphene oxide only, and graphene oxide and TSB only are our negative controls, and wild type MR-1 is the positive control to which all our transformed strains are compared. The O.D.600 measurements obtained from these experiments reflect both bacterial absorbance as well as reduced graphene oxide absorbance. So, the absolute O.D.600 due to reduced graphene oxide is obtained by correcting for O.D.600 of graphene oxide and TSB only and bacteria and TSB only.
Figure 1 shows the microbial reduction of graphene oxide with the transformed strain, cymA. It shows that transformation of S. oneidensis MR-1 with cymA still allows for reduction at a rate comparable to that of the wildtype. This figure represents the combined O.D.600 measurements for the reduced graphene oxide absorbance and bacterial absorbance.
The bacterial growth over the 48-hour period was measured by means of O.D.600 to determine the impact of cyma expression on bacterial growth. Figure 2 shows that bacteria expressing the cyma gene (black) had less overall growth over the 48-hour period and showed a more variable growth curve as compared to the wildtype (dark gray) and the other expressed genes.
To get the absolute O.D.600, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.600 were corrected for the O.D.600 for bacterial growth in TSB only. Figure 3 shows that after correction for bacterial growth cyma had a comparable rate of reduction with the wildtype where it appeared to be slightly slower.
Since cyma showed slower growth than wild-type MR-1 (Figure 2), we hypothesized that the induced gene may have been slightly toxic to the cells. We then observed the growth curve when the concentration of IPTG was decreased to 1.0mM to reduce the amount of cyma expression.
Figure 4 shows that with 1.0mM IPTG induction, microbial reduction of graphene oxide with the transformed strain cyma had comparable reduction rates to wild-type MR-1 and the empty vector, pcD8. From the figure it is seen that the rate of reduction of cyma without corrections for bacterial growth is slightly greater than that of the wildtype.
Figure 5 shows that there was no significant alteration in the growth for cyma for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2).
Figure 6 shows that with the correction for bacterial growth, microbial reduction with cyma with 1.0mM IPTG induction had a similar rate of reduction to the wildtype and a faster rate of reduction than pcD8 for the first 5 hours.
We carried out an induction time of 5 hours prior to reduction to allow cyma to already have the biofilm production fully active before we began the reduction reactions expression. This was carried out with either 1.5mM or 0.75mM of IPTG under the same conditions of shaking at 200 rpm.
From Figure 7, the microbial reduction of graphene oxide with the transformed strain cyma shows a greater rate of reduction to that of the wildtype without any corrections to the O.D.600 measurements for bacterial growth. It also shows a comparable rate of reduction to that of pcD8.
The bacterial growth measured by O.D.600 in Figure 8 shows that bacteria expressing the cyma gene (black) still had less overall growth over the 48-hour period and more variability in its growth curve as compared to the wildtype and the other expressed genes.
After corrections for bacterial growth, Figure 9 shows that cyma had the fastest rate of microbial reduction as compared to the wildtype and pcD8.
Figure 10 shows that the microbial reduction of graphene oxide with the transformed strain cyma at 0.75mM IPTG 5 hr induction has a greater rate of reduction than that of the wildtype and pcD8 without any corrections to the O.D.600 measurements for bacterial growth.
Figure 11, shows that the delay in growth in cyma (green) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of cyma still lags behind the wildtype and pcD8.
Figure 11, shows that the delay in growth in cyma (green) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of cyma still lags behind the wildtype and pcD8.
The growth curves of cyma show stochastic patterns for each of the IPTG conditions (Figure 2,5,8,11). It is thought that such patterns are observed due to the production of biofilm where increased biofilm production may cause bacteria to adhere to the sides of the plate wells, reducing the amount of cyma cells in the TSB for O.D.600 measurements.
The slopes at the steepest point on the curves were obtained for each induction condition to give the maximum reduction rate measured during the reduction period, and this was compared to that of wild-type MR-1.
The results show that after a 5hr induction with 1.5mM IPTG as well as 0.75mM IPTG, cyma has a faster rate of reduction as compared to wild-type MR-1 (Figure 9,12). Figure 13 shows that the maximum rate obtained for cyma at these conditions were 8.836 hr-1 and 8.468 hr-1, respectively, whereas the wildtype had a rate of 7.732 hr-1. There are comparable reduction rates with 1.5mM and 1.0mM IPTG for reduction immediately following induction (Figure 3,6). The reduction with cyma appears to be more similar to wild-type MR-1 at the lower IPTG concentration, 1.0mM (Figure 7).
The negative controls (TSB only, GO only, and GO and TSB only) show an insignificant change in O.D.600 over time indicating that the bacteria are responsible for the increase in O.D.600, that is, reduction.
Raman spectroscopy
Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by cyma over the 48 hour period.
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.
[7]Lehner, B. A.; Janssen, V. A.; Spiesz, E. M.; Benz, D.; Brouns, S. J.; Meyer, A. S.; van der Zant, H. S. Creation of Conductive Graphene Materials by Bacterial Reduction Using Shewanella Oneidensis. ChemistryOpen 2019, 8, 888–895.
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